http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Nakayama2012.igem.org - User contributions [en]2024-03-28T11:22:01ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T03:56:28Z<p>Nakayama: /* 4-4 Optimization of the best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
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2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
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3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
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4.</div><br />
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=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
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==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
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We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
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[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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==4-2 Confirmation of P(3HB) accumulated in cells==<br />
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To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
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[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
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==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
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==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
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Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
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[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
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[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
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The culture result is shown in Fig. 2-2-4-4-4.<br />
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[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
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*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
<br />
The results showed that TB medium was much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C was worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C didn’t make a significant difference. Even if there was no glucose, <I>E.coli</I> synthesized polymer (condition 9 & 10). We think that TB medium had glycerol and a lot of yeast extra, and then <I>E.coli</I> might have used them as carbon sources. In addition, the comparison of condition 4 & 5 indicates PA-Ca was not used as carbon sources. LB medium didn’t contain many carbon sources, so <I>E.coli</I> synthesized little polymer. In this case, adding PA-Ca didn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step was the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca. (the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29. Protocol]]<br />
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5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
<br />
<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol(2006), 24:1257-62</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/AttributionsTeam:Tokyo Tech/Attributions2012-10-27T03:41:51Z<p>Nakayama: /* Contribution */</p>
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Attributions and Contribution</div><br />
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__NOTOC__<br />
<br />
<br />
=Attribution=<br />
<br />
We are proud to say that all of the ideas we worked on this summer were proposed by undergraduate members of our own team. We also want to thank all those advisors and instructors who helped us discuss these ideas and guided us through doing the experiments. The whole experimental data produced by the Tokyo Tech 2012 team comes from experiments carried out all by undergraduates. <br />
<br />
The idea of "Romeo and juliet",the idea of "Cell-cell communication"(Takuo Sugiyama)and the idea of "PHA production"(Taku Nakayama) were all proposed by undergraduate members of our team. Modeling related to "Romeo and Juliet" was also done by undergraduate students (Nobuaki Yasuo).<br />
<br />
<br />
<br />
=Contribution=<br />
We thank J. Collins for providing JM2.300.<br />
<br />
We thank Prof. Ohta H for using fluorescence microscope.<br />
<br />
We thank iGEM HokkaidoU Japan 2012 for exchanging opinions about P(3HB) Production.<br />
<br />
We thank Prof. Konagaya A and Yoshida K for advising about Modeling.</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/AttributionsTeam:Tokyo Tech/Attributions2012-10-27T03:41:34Z<p>Nakayama: /* Contribution */</p>
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Attributions and Contribution</div><br />
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__NOTOC__<br />
<br />
<br />
=Attribution=<br />
<br />
We are proud to say that all of the ideas we worked on this summer were proposed by undergraduate members of our own team. We also want to thank all those advisors and instructors who helped us discuss these ideas and guided us through doing the experiments. The whole experimental data produced by the Tokyo Tech 2012 team comes from experiments carried out all by undergraduates. <br />
<br />
The idea of "Romeo and juliet",the idea of "Cell-cell communication"(Takuo Sugiyama)and the idea of "PHA production"(Taku Nakayama) were all proposed by undergraduate members of our team. Modeling related to "Romeo and Juliet" was also done by undergraduate students (Nobuaki Yasuo).<br />
<br />
=Contribution=<br />
We thank J. Collins for providing JM2.300.<br />
<br />
We thank Prof. Ohta H for using fluorescence microscope.<br />
<br />
We thank iGEM HokkaidoU Japan 2012 for exchanging opinions about P(3HB) Production.<br />
<br />
We thank Prof. Konagaya A and Yoshida K for advising about Modeling.</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T03:40:57Z<p>Nakayama: /* Reference */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
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<br><br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
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4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
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==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
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We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
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[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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==4-2 Confirmation of P(3HB) accumulated in cells==<br />
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To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
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[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
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==4-3 Confirmation of P(3HB) by GC/MS==<br />
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We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
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==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
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To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
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[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
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Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
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[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
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[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
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The culture result is shown in Fig. 2-2-4-4-4.<br />
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[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
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*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
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*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
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*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
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The results show that TB medium is much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesizes the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so <I>E.coli</I> synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29. Protocol]]<br />
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5.</div><br />
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=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
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We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
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[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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6.</div><br />
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=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol(2006), 24:1257-62</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ProjectTeam:Tokyo Tech/Project2012-10-27T03:36:18Z<p>Nakayama: /* Reference */</p>
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cell-cell communication </div><br />
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1.</div><br />
=Abstract=<br />
We designed a cell-cell communication system that makes two types of engineered <I>E.coli</I> play “Romeo and Juliet”. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI enzyme in Romeo cell, and 3OC12HSL is synthesized by LasI enzyme in Juliet cell. To reproduce the four scenes, we designed three subsystems: positive feedback system, band detect system, and communication-inverter dependent suicide system.<br />
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For scene 1 “Fall in love”, we achieved complete positive feedback system. First, we constructed and characterized two new Biobrick parts Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]). We confirmed that the cells containing Plux-LasI (Plux-LasI cell) produced 3OC12HSL in response to 3OC6HSL induction (Fig2-1-1-2) and Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction (Fig2-1-1-3). Second, by co-culturing these two types of signal producer cells, we then confirmed complete positive feedback system where the production of a signal activates the production of the other signal. Red arrows & blue arrows in the Fig2-1-1-4 strongly suggest that our positive feedback system worked accurately. Finally, to further confirm our positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. When both Plux-LasI cell and Plas-LuxI cell coexist, the result shows that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL (Fig2-1-1-1, 0-1h), whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level (Fig2-1-1-1, 1-2h). This behavior strongly suggests the appearance of the positive feedback. We think this is the most important result in our project.<br />
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For scene 2 “Juliet’s deathlike sleep”, we designed the 3OC6HSL-dependent band detect system. For this system, we characterized Plux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]), which is important part for our band detect system. Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) showed fluorescence only in the presence of both 3OC6HSL and aTc. In the presence of both inducers, the culture showed about 200-fold higher fluorescence intensity than that in the absence of both inducers (Fig2-1-1-5).<br />
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For scene 3 “Romeo’s suicide” and Scene4 “Juliet’s suicide”, we constructed two communication inverters: Plux-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) and Plas-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]). Plux-LacI expresses LacI repressor in the presence of 3OC6HSL. On the other hand, Plas-LacI expresses LacI repressor in the presence of 3OC12HSL. <br />
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Moreover, we conducted a simulation to confirm the feasibility of our cell-cell communication system.<br />
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[[File:positivefeedbackassay20tokyotech.png|700px|thumb|center|Fig2-1-1-1,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay.7ETime-dependent_change_assay.7E Time-dependent change assay]]]]<br />
[[File:positivefeedbackassay18tokyotech.png|170px|thumb|left|Fig2-1-1-2,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC6HSL-dependent_3OC12HSL_production_module Go to <br>"Construction_of<br>the_3OC6HSL-dependent<br>3OC12HSL_production"]]]]<br />
[[File:positivefeedbackassay19tokyotech.png|170px|thumb|left|Fig2-1-1-3,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC12HSL-dependent_3OC6HSL_production_module Go to <br>"Construction_of<br>the_3OC12HSL-dependent<br>3OC6HSL_production"]]]]<br />
[[File:positivefeedbackassay30tokyotech.png|150px|thumb|left|Fig2-1-1-4,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay_.7ECo-culture_assay.7E Go to <br>"Co-culture assay"]]]]<br />
[[File:positivefeedbackassay80tokyotech.png|150px|thumb|left|Fig2-1-1-5,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to <br>"Band detect system"]]]]<br />
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2.</div><br />
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=Story=<br />
We make our cute E.coli play “Romeo and Juliet” which is one of Shakespeare’s most famous plays. In this project, we define the signal that E.coli produce as the romantic feeling of Romeo and Juliet. In this project, we will recreate the love story of "Romeo & Juliet", by using "Cell-cell communication"<br />
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[[File:tokyotechStory.png|500px|thumb|right|Fig2-1-2-1, the story of “Romeo and Juliet"]]<br />
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The story that we reproduce is divided into four scenes.(Fig2-1-2-1)<br />
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'''(Scene 1)''' Romeo meets and falls in love with Juliet. Once the love between two people stimulates each other, they become deeply attached and cannot live without each other.<br />
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'''(Scene 2)''' However, Juliet knows that their love will not be accepted by society because of family feud. To keep their relationship, Juliet plans to pretend to be dead. She takes a sleeping potion that makes her fall into a deathlike sleep. <br />
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'''(Scene 3)''' Romeo has heard of Juliet’s death without knowing the fact that Juliet is alive. Romeo decides to commit suicide by taking poison in response to Juliet’s “deathlike sleep”. <br />
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'''(Scene 4)''' Juliet awakes to find Romeo dead beside her. She decides to commit suicide in response to Romeo’s suicide. She stabs herself with a dirk.<br />
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Design of genetic circuit</div><br />
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|video1 Behavior of our circuit for “Romeo and Juliet”<br />
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[[File:gene circuit tokyotech.png|400px|thumb|left|Fig2-1-2-2, Circuit design for “Romeo and Juliet”]]<br />
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Pon: promoter which is turned on.<br />
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Plux: promoter activated by LuxR/3OC6HSL complex.<br />
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Plas: promoter activated by LasR/3OC12HSL complex.<br />
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Plac: promoter repressed by LacI.<br />
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Plux/tet: hybrid promoter activated by LuxR/3OC6HSL complex and repressed by TetR<br />
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We designed a cell-cell communication system that makes <I>E.col</I>i play “Romeo and Juliet”. (Video1) The cell-cell communication system is composed of two types of engineered <I>E.coli</I> each of which plays Romeo and Juliet, respectively. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI enzyme in Romeo cell, and 3OC12HSL is synthesized by LasI enzyme in Juliet cell. To reproduce the four scenes, we designed three subsystems: positive feedback system, band detect system, and communication-inverter dependent suicide system.<br />
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'''First''', to represent “Scene1 Fall in love”, we designed a positive feedback system in which the production of a signal activates the production of the other signal. <br />
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'''Second''', we applied a 3OC6HSL-dependent band detect system to represent “Scene2 Juliet’s deathlike sleep” by a stop of 3OC12HSL production. When the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR is enough level to repress the expression of LasI. <br />
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'''Third''', to realize “Scene3 Romeo’s suicide” in response to Juliet’s deathlike sleep, we designed communication –inverter dependent suicide system in Romeo cell.<br />
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'''Finally''', to realize “Scene4 Juliet’s suicide” in response to Romeo’s suicide, we also designed communication-inverter dependent suicide system in Juliet cell.<br />
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Scene 1 Fall in love: positive feedback system</div><br />
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[[File:gene circuit withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-3, Scene 1 Fall in love: positive feedback system]]<br />
The love between Romeo and Juliet is realized by positive feedback of cell-cell communication signals (Fig2-1-2-3). In our positive feedback system, two types of <I>E.coli</I> communicate and regulate each other’s production of signal. An increase of one signal causes the increase of the other signal production, resulting in the increase of the signal itself. For the positive feedback system, two quorum sensing modules, LuxI/LuxR and LasI/LasR, which enable two-way communication, were used. Juliet cell expresses LuxR, which is a 3OC6HSL-dependent transcriptional regulator, constitutively. While Romeo cell expresses LasR, which is a 3OC12HSL-dependent transcriptional regulator, constitutively.<br />
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Gene activations in the positive feedback proceed in the following manner. In Juliet cell, 3OC6HSL is bound by LuxR to form LuxR-3OC6HSL complex. The complex activates the expression of LasI. The accumulation of LasI, 3OC12HSL-production enzyme, increases concentration of the signaling molecule in the medium. In the mean time, LasR-3OC12HSL complex in the Romeo cell similarly activates the expression of LuxI, which produce 3OC6HSL. Thus the concentration of both signal molecules in the medium becomes higher than that in initial conditions. The higher concentration of signal molecules, the more complexes exist in each cell. Then the activation of LuxI and LasI gets stronger. As a result, the increase of the signal synthesis is accelerated.<br />
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Scene2 Juliet’s deathlike sleep: 3OC6HSL-dependent band detect system</div><br />
[[File:gene circuit band detect system withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-4, Scene 2 Juliet’s deathlike sleep: 3OC6HSL-dependent band detect system]]<br />
The “Juliet’s deathlike sleep”, which is represented by the stop of 3OC12HSL production in a high concentration of Romeo signal 3OC6HSL, is realized by 3OC6HSL-dependent band detect system (Fig2-1-2-4). The band detect system is composed of Plux promoter, new Plux/tet hybrid promoter, and two regulator proteins. One of the proteins, LuxR, is expressed constitutively. The other one, TetR, is under Plux promoter, which is regulated by LuxR-3OC6HSL complex. The target gene of the band detect system is under Plux/tet hybrid promoter, which is regulated by LuxR-3OC6HSL complex and TetR.<br />
When the concentration of 3OC6HSL reaches the moderate level, LuxR-3OC6HSL complex moderately activates the expression of TetR and LasI. In this situation, the concentration of TetR is not enough to repress the expression of LasI. Thus, Juliet cell produces 3OC12HSL. As the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR reaches to enough level to repress LasI expression. As a result, 3OC12HSL production by Juliet cell is stopped though Juliet cell is alive.<br />
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Scene3 Romeo’s suicide: communication –inverter dependent suicide system in Romeo cell</div><br />
[[File:gene circuit suicide withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-5, Scene3 Romeo’s suicide: communication –inverter dependent suicide system in Romeo cell]]<br />
“Romeo’s suicide” in response to Juliet’s deathlike sleep is realized by communication-inverter dependent suicide system in Romeo cell (Fig2-1-2-5). In the presence of 3OC12HSL, LacI whose expression is regulated by LasR-3OC12HSL complex represses the expression of a lysis gene. Thus, Romeo cell keeps alive when Juliet cell produces 3OC12HSL. However, when Juliet cell is in deathlike sleep, supply of 3OC12HSL is stopped. Then the expression of LacI is also stopped in Romeo cell. In the absence of LacI, the lysis gene is expressed and Romeo cell dies.<br />
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Scene4 Juliet’s suicide: communication-inverter dependent suicide system in Juliet cell</div><br />
[[File:gene circuit juliet suicide withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-6, Scene4 Juliet’s suicide: communication-inverter dependent suicide system in Juliet cell]] <br />
“Juliet’s suicide” in response to Romemo’s suicide is realized by communication-inverter dependent suicide system in Juliet cell (Fig2-1-2-6). Juliet cell can keep alive in the presence of 3OC6HSL, because the lysis gene is repressed by LacI whose expression is regulated by LuxR-3OC6HSL complex. After the death of Romeo cell, supply of 3OC6HSL is stopped, so the expression of LacI is stopped. As a result, lysis gene is expressed and Juliet cell dies.<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Assays=<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3-1.</div><br />
<br />
==Assays for Positive feedback system ==<br />
[[File:positivefeedbackassay17tokyotech.png|300px|thumb|right| Fig2-1-3-1-1, Positive feedback system in the cell-cell communication]]<br />
===Introduction of the positive feedback===<br />
<br><br><br><br />
<br />
The positive feedback in the Romeo and Juliet cell-cell communication system is composed of two types of the engineered cells, 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) and 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell). In this positive feedback system, 3OC6HSL produced by Plas-LuxI cell activates the production of 3OC12HSL by Plux-LasI cell, and vice versa. For the implementation of the positive feedback system, several new Biobrick parts are required (Fig2-1-3-1-1).<br />
<br><br><br><br><br><br><br><br><br><br><br />
===Construction of the 3OC6HSL-dependent 3OC12HSL production module===<br />
<br />
<br />
For construction of the 3OC6HSL-dependent 3OC12HSL production module, we firstly constructed a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]). Plux-LasI cell is an engineered <I>E.coli</I> that contains a 3OC6HSL-dependent LasI generator and a constitutive LuxR generator. As a 3OC6HSL-dependent LasI generator, we constructed a new Biobrick part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) by combining Plux promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) and LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]<br />
). As a constitutive LuxR generator, we used Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]). By introducing Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
) and Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]) into <I>E.coli</I> strain JM2.300, we constructed Plux-LasI cell.<br />
Then we performed a reporter assay by using Las reporter cell to characterize the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022])<br />
. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell (ΔP-LasI cell) that contains, in addition to Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]), promoterless-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]) <br />
instead of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) (Fig2-1-3-1-2). <br />
[[File:positivefeedbackassay21tokyotech.png|550px|thumb|center|Fig2-1-3-1-2, Las sender strain and Las reporter strain]]<br />
<br><br />
The ⊿P-LasI cell does not produce 3OC12HSL even though 3OC6HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-3).<br />
<br />
[[File:positivefeedbackassay22tokyotech.png|500px|thumb|center|Fig2-1-3-1-3, How to perform 3OC6HSL-dependent 3OC12HSL production assay]]<br />
<br><br><br />
We prepared four conditions as follow.<br />
<br />
A) Culture containing Plux-LasI cell without 3OC6HSL induction<br />
<br />
B) Culture containing Plux-LasI cell with 3OC6HSL induction<br />
<br />
C) Culture containing ⊿P-LasI cell without 3OC6HSL induction<br />
<br />
D) Culture containing ⊿P-LasI cell with 3OC6HSL induction<br />
<br />
Using the supernatant of the four culture conditions, we performed the reporter assay. <br />
<br />
In the reporter assay, we used a Las reporter strain that contains Ptrc-LasR and Plas-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649001 BBa_K649001]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.<br />
<br><br><br />
[[File:positivefeedbackassay23tokyotech.png|450px|thumb|right|Fig2-1-3-1-4, 3OC6HSL-dependent 3OC12HSL production]]<br />
Fig2-1-3-1-4 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the Las reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
). From this experiment, we confirmed that a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
) synthesized enough concentration of 3OC12HSL to induce the Las reporter cell. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/C6#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br />
<br />
===Construction of the 3OC12HSL-dependent 3OC6HSL production module===<br />
<br />
For construction of the 3OC12HSL-dependent 3OC6HSL production module, we firstly constructed a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]<br />
). Plas-LuxI cell is an engineered <I>E.coli</I> that contains a 3OC12HSL-dependent LuxI generator and a constitutive LasR generator. As the 3OC12HSL-dependent LuxI generator, we constructed a new Biobrick part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]<br />
)by combining Plas promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000]<br />
). and LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008]<br />
). As a constitutive LasR generator, we used Ptrc-LasR. By introducing Plas-LuxI and Ptrc-LasR into <I>E.coli</I> strain JM 2.300, we constructed Plas-LuxI cell.<br />
Then we performed a reporter assay by using Lux reporter cell to characterize the function of Plas-LuxI. As the negative control of 3OC6HSL production, we prepared 3OC6HSL non-producer cell (ΔP-LuxI cell) that contains, in addition to Ptrc-LasR, promoterless-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008]) instead of Plas-LuxI (Fig2-1-3-1-5).<br />
[[File:positivefeedbackassay24tokyotech.png|550px|thumb|center|Fig2-1-3-1-5, Lux sender strain and Lux reporter strain]]<br />
<br />
<br />
The ΔP-LuxI cell does not produce 3OC6HSL even though 3OC12HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-6).<br />
[[File:positivefeedbackassay25tokyotech.png|500px|thumb|center|Fig2-1-3-1-6, How to perform 3OC12HSL-dependent 3OC6HSL production assay]]<br />
<br><br><br />
We prepared four conditions as follow.<br />
<br />
E) Culture containing Plas-LuxI cell without 3OC12HSL induction<br />
<br />
F) Culture containing Plas-LuxI cell with 3OC12HSL induction<br />
<br />
G) Culture containing ⊿P-LuxI cell without 3OC12HSL induction<br />
<br />
H) Culture containing ⊿P-LuxI cell with 3OC12HSL induction<br />
<br />
Using the supernatant of the four culture conditions, we performed the reporter assay. <br />
In the reporter assay, we used a Lux reporter strain that contains Ptet-LuxR and Plux-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395100 BBa_K395100]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.<br />
<br><br><br />
[[File:positivefeedbackassay26tokyotech.png|450px|thumb|right|Fig2-1-3-1-7, 3OC12HSL-dependent 3OC6HSL production]]<br />
<br />
Fig2-1-3-1-7 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) From this experiment, we confirmed that a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/C12#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br />
<br />
===Positive feedback assay ~Co-culture assay~===<br />
[[File:positivefeedbackassay17tokyotech.png|200px|thumb|right|Fig2-1-3-1-1, Positive feedback system in the cell-cell communication]]<br />
<br><br><br />
To accomplish complete positive feedback system, we mixed and co-cultured Plux-LasI cell and Plas-LuxI cell (Fig2-1-3-1-1). As a control co-culture, ⊿P-LasI cell and ⊿P-LuxI cell were mixed. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5nM) or 3OC12HSL (2.5nM) to the co-cultures. Including no-induction controls, we thus prepared six conditions (Fig2-1-3-1-8). To confirm that both 3OC6HSL and 3OC12HSL are produced in the positive feedback, the signals content in the supernatants of the co-cultures were evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-9).<br />
<br />
[[File:positivefeedbackassay27tokyotech.png|700px|thumb|center|Fig2-1-3-1-8, Six conditions prepared for positive feedback assay<br />
]]<br />
[[File:positivefeedbackassay28tokyotech.png|500px|thumb|center|Fig2-1-3-1-9, How to evaluate the positive feedback<br />
]]<br />
In these co-cultures, following behavior would be expected. In the condition I, 3OC6HSL induces 3OC12HSL production of Plux-LasI cell. Then, 3OC12HSL synthesized by Plux-LasI cell induces Plas-LuxI cell. The concentration of 3OC6HSL thus increases compared to the initial amount. Increased amount of 3OC6HSL induces higher production of 3OC12HSL. As a result, the production of both signals increased in the condition I. In this situation, the concentration of 3OC6HSL is higher than the initial concentration of 3OC6HSL (ideal positive feedback behavior). Similarly in the condition II, total amount of 3OC12HSL also increases compared to the initial amount. In the conditions III and VI, no signal is produced because of the lack of inducer. In conditions IV and V, little amount of 3OC6HSL and 3OC12HSL are remained without degradation, respectively.<br />
<br><br><br><br />
[[File:positivefeedbackassay31tokyotech.png|550px|thumb|right|Fig2-1-3-1-10, Positive feedback assay<br />
]]<br />
<br><br />
Fig2-1-3-1-10 shows that the fluorescence intensity of the Lux reporter cell in the condition I was higher than that in the condition IV. Similarly, the fluorescence intensity of the Las reporter in the condition II was higher than that in the condition V. <br />
From these results and circuit design described above, it is suggested that the appearance of positive feedback where cooperation of Plas-LuxI cell and Plux-LasI cell increase the production of 3OC6HSL and 3OC12HSL. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/positivefeedback1#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br><br><br />
<br><br><br><br />
<br />
===Positive feedback assay~Time-dependent change assay~===<br />
We confirmed the appearance of the positive feedback from the co-culture assay above. To further confirm the positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. <br />
To observe time-dependent change of the positive feedback, we prepared four conditions of co-culture : Plux-LasI cell and Plas-LuxI cell coexisted in condition i, ΔP-LasI cell and Plas-LuxI cell in condition ii, Plux-LasI cell and ΔP-LuxI cell in condition iii, and ΔP-LasI cell and ΔP-LuxI cell in condition iv. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5 nM) to each co-culture.<br />
<br />
<br><br />
[[File:positivefeedbackassay85tokyotech.png|700px|thumb|center|Fig2-1-3-1-11,Four conditions prepared for Time-dependent change assay]]<br />
<br><br />
To estimate the concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback, the signals content in the supernatants of the co-cultures were collected at the time of 0, 0.5, 1, 2, 4 hours. The concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback was evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-11). Fig2-1-3-1-11 also shows ideal signal production of the positive feedback in the cell-cell communication.<br />
<br><br />
[[File:positivefeedbackassay86tokyotech.png|700px|thumb|center|Fig2-1-3-1-12,Time-dependent change assay]]<br />
<br><br />
<br />
Fig2-1-3-1-12 shows the characterization of time-dependent signal increase in the positive feedback in the cell-cell communication. Solid lines represent the fluorescence intensities of Las reporters in the four conditions. Dotted lines represent the fluorescence intensities of Lux reporters in the four conditions.<br />
<br />
<br />
As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the fig shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.<br />
<br />
<br />
Fig2-1-3-1-12 also suggests that the advantage in signal production in the presence of both signal producers, Plux-LasI cell and Plas-LuxI cell. Only when Plux-LasI cell and Plas-LuxI cell were cultured together and allowed to communicate with one another, the fluorescence intensity of Las reporter and Lux reporter increased drastically. On the other hand, when either Plux-LasI cell or Plas-LuxI cell existed (condition ii and iii), drastic rearing in the GFP expression was not observed. Especially in the condition iii, GFP in the las reporter cell is expressed to some extent (about 2-folds lower than in condition i). This behavior shows that the production of 3OC12HSL in the condition iii cannot exceed the production of 3OC12HSL in the positive feedback state. This is because ΔP-LuxI cell cannot produce any 3OC6HSL to activate Plux-LasI cell in this condition, though initially added 3OC6HSL activated Plux-LasI cell and Plux-LasI cell produced 3OC12HSL to some extent. Moreover, it goes without saying that, the fluorescence intensity of both Las reporter and Lux reporter remained low level through observation in the absence of both signal producer cell. <br />
<br />
<br />
In addition, low concentration of 3OC6HSL was detected by the Lux reporter in the condition ii, iii & iv, in which 3OC6HSL is not produced. However, fluorescence intensity in these conditions decreased with time. Therefore, it is indicated that these GFP expressions were derived from initially added 3OC6HSL. <br />
<br />
<br />
From these overall results, we further confirmed the positive feedback in the cell-cell communication and characterized time-dependent change of the positive feedback. Only when the two types of signal producer cells were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increased as compared with other conditions.<br />
<br />
<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/positivefeedback2#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br />
<br />
===Conclusion of positive feedback system===<br />
In this study, we designed and implemented a positive feedback system in the cell-cell communication that is composed of the Plux-LasI cell and the Plas-LuxI cell. <br />
<br />
<br />
First, we confirmed that the Plux-LasI cell synthesized enough concentration of 3OC12HSL to induce the Las reporter cell and the Plas-LuxI cell synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell. In the process of the implementation, we constructed two new Biobrick parts Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]), the new Biobrick parts that can be regulated by the induction of 3OC6HSL and 3OC12HSL, respectively. <br />
<br />
By co-culturing of these two types of <I>E.coli</I>, we then confirmed that higher concentration of a signal than initial conditions was detected through production of the other signal. These results indicated appearance of the positive feedback. <br />
<br />
To further confirm the positive feedback system, we also characterized the time-dependent change of this positive feedback in the cell-cell communication. The result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly verifies the appearance of the positive feedback.<br />
<br />
Only when the two types of <I>E.coli</I> were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increase as compared to other conditions.<br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3-2.</div><br />
<br />
==Band detect system==<br />
===Introduction of band detect system===<br />
====the original band detect system====<br />
To achieve our goals, we need the 3OC6HSL-dependent band detect system.<br />
<br />
[[File:luxtethybrid20tokyotech.png|700px|thumb|center|Fig2-1-3-2-1, <br>(a)the band detect system constructed by Ron Weiss Lab. <br>(b)Our new band detect system]]<br />
<br />
In 2005, the first band detect system in synthetic biology was constructed by Ron Weiss Lab.([[#Reference|[1]]]<br />
) In that system (Fig2-1-3-2-1(a)), 3OC6HSL-LuxR complex activates the expression of LacIM1 repressor and CI repressor. Furthermore, CI repressor represses the expression of LacI. Then, both LacIM1 repressor and LacI repressor repress the expression of GFP. <br />
When the concentration of 3OC6HSL is high, the expression of LacIM1 is promoted strongly. As a result, the expression of GFP is repressed. When the concentration of 3OC6HSL is moderate, it results in the moderate level of CI and LacIM1. Because the repression efficiency of CI is sufficiently high, the LacI expression remains repressed. However, in this situation, the concentration of LacIM1, whose repression efficiency is low, is not sufficient to repress the GFP expression. Thus, GFP is expressed. When the concentration of 3OC6HSL is low, the expression of CI is only at low level. This enables the expression of LacI, then the expression of GFP is repressed again.<br />
In this way, the expression of GFP is activated by the band detect system under the particular range of the concentration of 3OC6HSL.<br />
<br />
In this project, we invented the new band detect system (Fig2-1-3-2-1(b)). Compared with the old one, our new band detect system has a merit, that is constructed with fewer components. By reducing components in cells, we can avoid the useless complexity and the inhibition of cell growth.<br />
We employed the lux/tet hybrid promoter in the system. In the following, we described the details of our system.<br />
<br />
====our new band detect system====<br />
The band detect system for cell-cell communication system is composed of lux/tet hybrid promoter,Plux-TetR,and Pon-LuxR. In contrast to the Weiss lab’s band detect system, which take advantage of difference in activity between wild-type repressor and its mutant, we used a hybrid promoter for the system. The expression of target gene, LasI, is regulated by both of LuxR-3OC6HSL complex and TetR through binding of them to lux/tet promoter.<br />
The expression of TetR from lux promoter is also regulated by LuxR-3OC6HSL (Fig2-1-3-2-2). Thus, concentration of 3OC6HSL affects both of lux/tet hybrid promoter and lux promoter since Pon-LuxR constitutively express LuxR. <br />
[[File:luxtethybrid12tokyotech.png|500px|thumb|center|Fig2-1-3-2-2]]<br />
When the concentration of 3OC6HSL is initial level, LuxR-3OC6HSL complex activates the expression of LasI. Though TetR is also expressed, its concentration is not enough to repress the expression of LasI.(Fig2-1-3-2-3)<br />
[[File:luxtethybrid1tokyotech.png|500px|thumb|center|Fig2-1-3-2-3]]<br />
As the concentration of 3OC6HSL increases to moderate level gradually by the positive feedback system, the expression of TetR and LasI also increases. In this situation, the concentration of TetR is still not enough to repress the expression of LasI.(Fig2-1-3-2-4)<br />
[[File:luxtethybrid2tokyotech.png|500px|thumb|center|Fig2-1-3-2-4]]<br />
<br />
When the concentration of 3OC6HSL is high level, the concentration of TetR reaches to the enough level to repress LasI expression(Fig2-1-3-2-5). For the implementation of the band detect system, lux/tet hybrid promoter is required. <br />
[[File:luxtethybrid3tokyotech.png|500px|thumb|center|Fig2-1-3-2-5]]<br />
The repression of production of 3OC12HSL results in arrest of the 3OC6HSL supply from Romeo cell. Then the LuxR-3OC6HSL complex cannot form and the expression of LasI is stopped.(Fig2-1-3-2-6)<br />
[[File:luxtethybrid4tokyotech.png|500px|thumb|center|Fig2-1-3-2-6]]<br />
<br />
===Result===<br />
[[File:luxtethybrid5tokyotech.png|450px|thumb|right|Fig2-1-3-2-7,lux/tet hybrid promoter assay]]<br />
For construction of the band detect system, we developed a new part lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]). The lux/tet hybrid promoter, which is composed of a LuxR operator and two TetR operators, activates the expression of the downstream gene only when LuxR-3OC6HSL complex exists and active TetR does not exist. To characterize the function of the lux/tet hybrid promoter, we constructed a part, Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) by inserting the promoter in front of a GFP coding sequence. By using the reporter cell that contains Plux/tet-GFP and constitutive LuxR and TetR generator (PlacIq-LuxR-Ptrc-TetR), we measured the fluorescence intensity of the reporter cell. In the measurement, we confirmed the GFP expression under the four different combinations of two inducers, 3OC6HSL and aTc (anhydrous tetracycline). In the absence of the both inducers, the culture with lux-tet hybrid promoter-gfp showed the background–fluorescence intensity generated by promoterless-rbs-gfp on pSB3K3. The presence of either 3OC6HSL or aTc alone had little effect on increasing the fluorescence intensity. In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers(Fig2-1-3-2-7). This result confirmed that the assembly of the LuxR operator and the two TetR operators integrated the inputs of 3OCH6HSL and aTc into the output of GFP transcription. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/banddetect#Materials_.26_Method Materials and Methods]]<br />
<br />
===Discussion===<br />
[[File:luxtethybrid31tokyotech.png|400px|thumb|right|Fig2-1-3-2-8]]<br />
[[File:luxtethybrid32tokyotech.png|400px|thumb|right|Fig2-1-3-2-9]]<br />
<br />
In this study, we improved lux/tet hybrid promoter parts by developing a new lux/tet hybrid promoter that is regulated by LuxR-3OC6HSL and TetR. Even though a former team had reported a lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K176078 BBa_K176078]), their assay did not contain the inducer combination:3OC6HSL(-) and aTc(+).Thus, data of lux/tet hybrid promoter in Biobrick Registry was not sufficient(Fig2-1-3-2-9). In contrast, all four combinations were confirmed in our new Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025])<br />
Therefore, our work is considered as the improvement of the lux/tet hybrid promoter.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br />
<br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3-3.</div><br />
<br />
==communication-inverter dependent suicide system==<br />
<br />
Introduction:<br />
<br />
In order to reproduce the suicides of Romeo & Juliet’s, the communication dependent inverter system that express lysis protein only when the signals do not exist is required.To realize the communication dependent inverter system, we constructed Plas-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) and Plux-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) that express LacI to repress the target gene only when LasR-3OC12HSL and LuxR-3OC6HSL exist respectively.<br />
<br />
<br />
'''3OC12HSL dependent'''<br />
<br />
construction<br />
<br />
We constructed a 3OC12HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) by ligating PlasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).<br />
<br />
'''3OC6HSL dependent'''<br />
<br />
construction<br />
<br />
We constructed a 3OC6HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) by ligating Plux ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).<br />
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4.</div><br />
<br />
=Modeling=<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by wet experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling#Model_development Detailed descriptions for Modeling development]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|500px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4, the story of “Romeo and Juliet”]]<br />
<br><br />
'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
<br />
'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
<br />
'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
<br />
'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
<br />
<br><br><br><br><br><br />
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==Result2: Validation of three subsystems’ function==<br />
<br />
In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
<br />
<br />
'''(1)Positive feedback system'''<br />
<br />
To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
<br />
<br />
[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increase. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
<br />
<br />
'''(2)Band detect system'''<br />
<br />
Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
<br />
[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.(Fig2-1-4-8)<br />
<br />
<br />
[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
<br><br><br><br><br><br><br><br />
On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration(Fig2-1-4-9). However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
<br />
<br><br><br><br><br><br><br />
<br />
In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
<br><br><br />
'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,<br>(a)the concentration of Juliet signals <br>(b)the concentration of lysis proteins in Romeo cells <br>(c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
<br><br><br><br><br />
'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gene) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-11, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
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==a==<br />
<br />
[[File:tokyotechMode7ing1.png|600px|thumb|center|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
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[[File:tokyotechMode7ing2.png|600px|thumb|center|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
<br />
[[File:tokyotechMode7ing3.png|600px|thumb|center|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing4.png|600px|thumb|center|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
<br />
[[File:tokyotechMode7ing5.png|600px|thumb|center|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
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5.</div><br />
<br />
=Application=<br />
In this project, we constructed the new cell-cell communication system. The special features of our system are as follows.<br />
<br />
1. One kind of signal activates the production of another one via the positive feedback system.<br />
<br />
2. The band detect system regulates the excessive production of LasI enzymes.<br />
<br />
3. Communication-inverter dependent suicide system controls the cell population.<br />
<br />
By applying these features, we would achieve a new system for manufacturing. <br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
<br />
(1) Sustainable manufacturing</div><br />
[[File:Application5tokyotech.png|400px|thumb|right|Fig2-1-5-1, the circuit with positive feedback system and band detect system]]<br />
<br />
Simulation shows that the collaboration of positive feedback system and band detect system (Fig2-1-5-1) produces the signals at the moderate level (the yellow line and the purple line in Fig2-1-5-2). <br />
[[File:Application85tokyotech.png|800px|thumb|center|Fig2-1-5-2, the time-dependent change of 3OC12HSL concentration in four situations]]<br />
<br />
Fig2-1-5-2 shows the time-dependent change of 3OC12HSL concentration in the culture. In the collaboration of positive feedback system and band detect system, the concentration of 3OC12HSL settles into a moderate steady state (the yellow line or the purple line), compared with high concentration in the simple positive feedback system (the red line). When two types of E.coli are cultured separately, 3OC12HSL is not produced (the blue line). <br />
In addition, by changing parameters, we can control the concentration in the final steady state (the yellow line and the purple line).<br />
<br />
We propose inserting the coding region of desired proteins to downstream region of LuxI or LasI in this system. In that case, the production of desired proteins is activated only when two types of E.coli are co-cultured. Then, once it starts, the increase of production is accelerated by the positive feedback system. Furthermore, the band detect system plays an important role. The band detect system regulates the excessive production of proteins in the host cells. Thereby, it is expected that the host cells can avoid growth inhibition caused by metabolic imbalance.<br />
<br />
As described above, by applying this system, we would lighten the metabolic burdens on the host cells and achieve sustainable manufacturing system. Moreover, it is suggested that we can control the concentration in the final steady state.<br />
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(2) Division of labor</div><br />
It is known that microbial consortia can perform even more complicated tasks through the division of labor than individual strains. For example, when the products require multiple steps to convert the substrates, the system with multiple strains which is dedicated to each step has two advantages, compared to the system with a single strain. First, by limiting the number of exogenous elements in the host cells, metabolic imbalance in the cells reduces. Second, it is possible to improve the reaction efficiency by isolating the engineered circuits dedicated to each reaction. <br />
<br />
In our system, the two types of E.coli communicate with each other with signal molecules and control the expression of proteins mutually. By applying this system, we would achieve more efficient manufacturing, for example, the simultaneous conversion of sugar mixtures at similar rates.<br />
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6.</div><br />
<br />
=Reference=<br />
<br />
1. Basu S, Gerchman Y, Collins CH, Arnold FH, & Weiss R (2005) A synthetic multicellular system for programmed pattern formation. Nature 434(7037):1130-1134.<br />
<br />
2. You L, Cox RS, Weiss R, & Arnold FH (2004) Programmed population control by cell-cell communication and regulated killing. Nature 428:868-871.<br />
<br />
3. Balagadde FK, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4:187<br />
<br />
4. J Biol Eng. (2008) co-fermentation strategy to consume sugar mixtures effectively Published online 2008 February 27. <br />
<br />
5. Brenner K, You L, Arnold FH Engineering microbial consortia: a new frontier in synthetic biology Trends in Biotechnology [2008, 26(9):483-489]<br />
<br />
7. Akihiko K, et al. (2011) Cluster Newton Method for Sampling Multiple Solutions of an Underdetermined Inverse Problem: Parameter Identification for Pharmacokinetics. National Institute of Informatics, NII-2011-002E.</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-27T03:28:34Z<p>Nakayama: /* Construction of pha-C1-A-B1 in Biobrick format */</p>
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=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==A. Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==B. Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
==C. Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D. Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
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<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesizes the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so <I>E.coli</I> synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9 & 10)[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29. Protocol]]<br />
<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization of the best culture condition to synthesize P(3HB).==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/File:Tokyotech_PHB4.pngFile:Tokyotech PHB4.png2012-10-27T03:20:25Z<p>Nakayama: uploaded a new version of &quot;File:Tokyotech PHB4.png&quot;</p>
<hr />
<div></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ProjectTeam:Tokyo Tech/Project2012-10-27T03:16:20Z<p>Nakayama: /* Result2: Validation of three subsystems’ function */</p>
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cell-cell communication </div><br />
</div class="whitebox"><br />
<div class="whitebox"><br />
__TOC__<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Abstract=<br />
We designed a cell-cell communication system that makes two types of engineered <I>E.coli</I> play “Romeo and Juliet”. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI enzyme in Romeo cell, and 3OC12HSL is synthesized by LasI enzyme in Juliet cell. To reproduce the four scenes, we designed three subsystems: positive feedback system, band detect system, and communication-inverter dependent suicide system.<br />
<br />
For scene 1 “Fall in love”, we achieved complete positive feedback system. First, we constructed and characterized two new Biobrick parts Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]). We confirmed that the cells containing Plux-LasI (Plux-LasI cell) produced 3OC12HSL in response to 3OC6HSL induction (Fig2-1-1-2) and Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction (Fig2-1-1-3). Second, by co-culturing these two types of signal producer cells, we then confirmed complete positive feedback system where the production of a signal activates the production of the other signal. Red arrows & blue arrows in the Fig2-1-1-4 strongly suggest that our positive feedback system worked accurately. Finally, to further confirm our positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. When both Plux-LasI cell and Plas-LuxI cell coexist, the result shows that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL (Fig2-1-1-1, 0-1h), whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level (Fig2-1-1-1, 1-2h). This behavior strongly suggests the appearance of the positive feedback. We think this is the most important result in our project.<br />
<br />
For scene 2 “Juliet’s deathlike sleep”, we designed the 3OC6HSL-dependent band detect system. For this system, we characterized Plux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]), which is important part for our band detect system. Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) showed fluorescence only in the presence of both 3OC6HSL and aTc. In the presence of both inducers, the culture showed about 200-fold higher fluorescence intensity than that in the absence of both inducers (Fig2-1-1-5).<br />
<br />
For scene 3 “Romeo’s suicide” and Scene4 “Juliet’s suicide”, we constructed two communication inverters: Plux-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) and Plas-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]). Plux-LacI expresses LacI repressor in the presence of 3OC6HSL. On the other hand, Plas-LacI expresses LacI repressor in the presence of 3OC12HSL. <br />
<br />
Moreover, we conducted a simulation to confirm the feasibility of our cell-cell communication system.<br />
<br />
<br />
[[File:positivefeedbackassay20tokyotech.png|700px|thumb|center|Fig2-1-1-1,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay.7ETime-dependent_change_assay.7E Time-dependent change assay]]]]<br />
[[File:positivefeedbackassay18tokyotech.png|170px|thumb|left|Fig2-1-1-2,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC6HSL-dependent_3OC12HSL_production_module Go to <br>"Construction_of<br>the_3OC6HSL-dependent<br>3OC12HSL_production"]]]]<br />
[[File:positivefeedbackassay19tokyotech.png|170px|thumb|left|Fig2-1-1-3,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC12HSL-dependent_3OC6HSL_production_module Go to <br>"Construction_of<br>the_3OC12HSL-dependent<br>3OC6HSL_production"]]]]<br />
[[File:positivefeedbackassay30tokyotech.png|150px|thumb|left|Fig2-1-1-4,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay_.7ECo-culture_assay.7E Go to <br>"Co-culture assay"]]]]<br />
[[File:positivefeedbackassay80tokyotech.png|150px|thumb|left|Fig2-1-1-5,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to <br>"Band detect system"]]]]<br />
<br />
<br><br><br><br><br><br><br />
<br><br><br><br />
</div><br />
</div><br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=Story=<br />
We make our cute E.coli play “Romeo and Juliet” which is one of Shakespeare’s most famous plays. In this project, we define the signal that E.coli produce as the romantic feeling of Romeo and Juliet. In this project, we will recreate the love story of "Romeo & Juliet", by using "Cell-cell communication"<br />
<br />
[[File:tokyotechStory.png|500px|thumb|right|Fig2-1-2-1, the story of “Romeo and Juliet"]]<br />
<br />
The story that we reproduce is divided into four scenes.(Fig2-1-2-1)<br />
<br />
'''(Scene 1)''' Romeo meets and falls in love with Juliet. Once the love between two people stimulates each other, they become deeply attached and cannot live without each other.<br />
<br />
'''(Scene 2)''' However, Juliet knows that their love will not be accepted by society because of family feud. To keep their relationship, Juliet plans to pretend to be dead. She takes a sleeping potion that makes her fall into a deathlike sleep. <br />
<br />
'''(Scene 3)''' Romeo has heard of Juliet’s death without knowing the fact that Juliet is alive. Romeo decides to commit suicide by taking poison in response to Juliet’s “deathlike sleep”. <br />
<br />
'''(Scene 4)''' Juliet awakes to find Romeo dead beside her. She decides to commit suicide in response to Romeo’s suicide. She stabs herself with a dirk.<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #191970; padding: 2px;"><br />
Design of genetic circuit</div><br />
<br />
{| style=text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"<br />
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|video1 Behavior of our circuit for “Romeo and Juliet”<br />
|-<br />
|align="center"|<html><iframe width="420" height="315" src="http://www.youtube.com/embed/bqhOHEFI65g" frameborder="0" allowfullscreen></iframe></html><br />
|}<br />
<br />
<br />
[[File:gene circuit tokyotech.png|400px|thumb|left|Fig2-1-2-2, Circuit design for “Romeo and Juliet”]]<br />
<br><br><br />
Pon: promoter which is turned on.<br />
<br />
Plux: promoter activated by LuxR/3OC6HSL complex.<br />
<br />
Plas: promoter activated by LasR/3OC12HSL complex.<br />
<br />
Plac: promoter repressed by LacI.<br />
<br />
Plux/tet: hybrid promoter activated by LuxR/3OC6HSL complex and repressed by TetR<br />
<br><br><br><br />
<br><br><br><br />
<br />
We designed a cell-cell communication system that makes <I>E.col</I>i play “Romeo and Juliet”. (Video1) The cell-cell communication system is composed of two types of engineered <I>E.coli</I> each of which plays Romeo and Juliet, respectively. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI enzyme in Romeo cell, and 3OC12HSL is synthesized by LasI enzyme in Juliet cell. To reproduce the four scenes, we designed three subsystems: positive feedback system, band detect system, and communication-inverter dependent suicide system.<br />
<br />
<br />
'''First''', to represent “Scene1 Fall in love”, we designed a positive feedback system in which the production of a signal activates the production of the other signal. <br />
<br />
'''Second''', we applied a 3OC6HSL-dependent band detect system to represent “Scene2 Juliet’s deathlike sleep” by a stop of 3OC12HSL production. When the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR is enough level to repress the expression of LasI. <br />
<br />
'''Third''', to realize “Scene3 Romeo’s suicide” in response to Juliet’s deathlike sleep, we designed communication –inverter dependent suicide system in Romeo cell.<br />
<br />
'''Finally''', to realize “Scene4 Juliet’s suicide” in response to Romeo’s suicide, we also designed communication-inverter dependent suicide system in Juliet cell.<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
Scene 1 Fall in love: positive feedback system</div><br />
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[[File:gene circuit withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-3, Scene 1 Fall in love: positive feedback system]]<br />
The love between Romeo and Juliet is realized by positive feedback of cell-cell communication signals (Fig2-1-2-3). In our positive feedback system, two types of <I>E.coli</I> communicate and regulate each other’s production of signal. An increase of one signal causes the increase of the other signal production, resulting in the increase of the signal itself. For the positive feedback system, two quorum sensing modules, LuxI/LuxR and LasI/LasR, which enable two-way communication, were used. Juliet cell expresses LuxR, which is a 3OC6HSL-dependent transcriptional regulator, constitutively. While Romeo cell expresses LasR, which is a 3OC12HSL-dependent transcriptional regulator, constitutively.<br />
<br />
Gene activations in the positive feedback proceed in the following manner. In Juliet cell, 3OC6HSL is bound by LuxR to form LuxR-3OC6HSL complex. The complex activates the expression of LasI. The accumulation of LasI, 3OC12HSL-production enzyme, increases concentration of the signaling molecule in the medium. In the mean time, LasR-3OC12HSL complex in the Romeo cell similarly activates the expression of LuxI, which produce 3OC6HSL. Thus the concentration of both signal molecules in the medium becomes higher than that in initial conditions. The higher concentration of signal molecules, the more complexes exist in each cell. Then the activation of LuxI and LasI gets stronger. As a result, the increase of the signal synthesis is accelerated.<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
Scene2 Juliet’s deathlike sleep: 3OC6HSL-dependent band detect system</div><br />
[[File:gene circuit band detect system withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-4, Scene 2 Juliet’s deathlike sleep: 3OC6HSL-dependent band detect system]]<br />
The “Juliet’s deathlike sleep”, which is represented by the stop of 3OC12HSL production in a high concentration of Romeo signal 3OC6HSL, is realized by 3OC6HSL-dependent band detect system (Fig2-1-2-4). The band detect system is composed of Plux promoter, new Plux/tet hybrid promoter, and two regulator proteins. One of the proteins, LuxR, is expressed constitutively. The other one, TetR, is under Plux promoter, which is regulated by LuxR-3OC6HSL complex. The target gene of the band detect system is under Plux/tet hybrid promoter, which is regulated by LuxR-3OC6HSL complex and TetR.<br />
When the concentration of 3OC6HSL reaches the moderate level, LuxR-3OC6HSL complex moderately activates the expression of TetR and LasI. In this situation, the concentration of TetR is not enough to repress the expression of LasI. Thus, Juliet cell produces 3OC12HSL. As the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR reaches to enough level to repress LasI expression. As a result, 3OC12HSL production by Juliet cell is stopped though Juliet cell is alive.<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
Scene3 Romeo’s suicide: communication –inverter dependent suicide system in Romeo cell</div><br />
[[File:gene circuit suicide withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-5, Scene3 Romeo’s suicide: communication –inverter dependent suicide system in Romeo cell]]<br />
“Romeo’s suicide” in response to Juliet’s deathlike sleep is realized by communication-inverter dependent suicide system in Romeo cell (Fig2-1-2-5). In the presence of 3OC12HSL, LacI whose expression is regulated by LasR-3OC12HSL complex represses the expression of a lysis gene. Thus, Romeo cell keeps alive when Juliet cell produces 3OC12HSL. However, when Juliet cell is in deathlike sleep, supply of 3OC12HSL is stopped. Then the expression of LacI is also stopped in Romeo cell. In the absence of LacI, the lysis gene is expressed and Romeo cell dies.<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
Scene4 Juliet’s suicide: communication-inverter dependent suicide system in Juliet cell</div><br />
[[File:gene circuit juliet suicide withpromoter tokyotech.png|400px|thumb|left|Fig2-1-2-6, Scene4 Juliet’s suicide: communication-inverter dependent suicide system in Juliet cell]] <br />
“Juliet’s suicide” in response to Romemo’s suicide is realized by communication-inverter dependent suicide system in Juliet cell (Fig2-1-2-6). Juliet cell can keep alive in the presence of 3OC6HSL, because the lysis gene is repressed by LacI whose expression is regulated by LuxR-3OC6HSL complex. After the death of Romeo cell, supply of 3OC6HSL is stopped, so the expression of LacI is stopped. As a result, lysis gene is expressed and Juliet cell dies.<br />
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3.</div><br />
<br />
=Assays=<br />
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3-1.</div><br />
<br />
==Assays for Positive feedback system ==<br />
[[File:positivefeedbackassay17tokyotech.png|300px|thumb|right| Fig2-1-3-1-1, Positive feedback system in the cell-cell communication]]<br />
===Introduction of the positive feedback===<br />
<br><br><br><br />
<br />
The positive feedback in the Romeo and Juliet cell-cell communication system is composed of two types of the engineered cells, 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) and 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell). In this positive feedback system, 3OC6HSL produced by Plas-LuxI cell activates the production of 3OC12HSL by Plux-LasI cell, and vice versa. For the implementation of the positive feedback system, several new Biobrick parts are required (Fig2-1-3-1-1).<br />
<br><br><br><br><br><br><br><br><br><br><br />
===Construction of the 3OC6HSL-dependent 3OC12HSL production module===<br />
<br />
<br />
For construction of the 3OC6HSL-dependent 3OC12HSL production module, we firstly constructed a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]). Plux-LasI cell is an engineered <I>E.coli</I> that contains a 3OC6HSL-dependent LasI generator and a constitutive LuxR generator. As a 3OC6HSL-dependent LasI generator, we constructed a new Biobrick part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) by combining Plux promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) and LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]<br />
). As a constitutive LuxR generator, we used Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]). By introducing Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
) and Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]) into <I>E.coli</I> strain JM2.300, we constructed Plux-LasI cell.<br />
Then we performed a reporter assay by using Las reporter cell to characterize the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022])<br />
. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell (ΔP-LasI cell) that contains, in addition to Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]), promoterless-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]) <br />
instead of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) (Fig2-1-3-1-2). <br />
[[File:positivefeedbackassay21tokyotech.png|550px|thumb|center|Fig2-1-3-1-2, Las sender strain and Las reporter strain]]<br />
<br><br />
The ⊿P-LasI cell does not produce 3OC12HSL even though 3OC6HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-3).<br />
<br />
[[File:positivefeedbackassay22tokyotech.png|500px|thumb|center|Fig2-1-3-1-3, How to perform 3OC6HSL-dependent 3OC12HSL production assay]]<br />
<br><br><br />
We prepared four conditions as follow.<br />
<br />
A) Culture containing Plux-LasI cell without 3OC6HSL induction<br />
<br />
B) Culture containing Plux-LasI cell with 3OC6HSL induction<br />
<br />
C) Culture containing ⊿P-LasI cell without 3OC6HSL induction<br />
<br />
D) Culture containing ⊿P-LasI cell with 3OC6HSL induction<br />
<br />
Using the supernatant of the four culture conditions, we performed the reporter assay. <br />
<br />
In the reporter assay, we used a Las reporter strain that contains Ptrc-LasR and Plas-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649001 BBa_K649001]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.<br />
<br><br><br />
[[File:positivefeedbackassay23tokyotech.png|450px|thumb|right|Fig2-1-3-1-4, 3OC6HSL-dependent 3OC12HSL production]]<br />
Fig2-1-3-1-4 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the Las reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
). From this experiment, we confirmed that a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]<br />
) synthesized enough concentration of 3OC12HSL to induce the Las reporter cell. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/C6#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br />
<br />
===Construction of the 3OC12HSL-dependent 3OC6HSL production module===<br />
<br />
For construction of the 3OC12HSL-dependent 3OC6HSL production module, we firstly constructed a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]<br />
). Plas-LuxI cell is an engineered <I>E.coli</I> that contains a 3OC12HSL-dependent LuxI generator and a constitutive LasR generator. As the 3OC12HSL-dependent LuxI generator, we constructed a new Biobrick part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]<br />
)by combining Plas promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000]<br />
). and LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008]<br />
). As a constitutive LasR generator, we used Ptrc-LasR. By introducing Plas-LuxI and Ptrc-LasR into <I>E.coli</I> strain JM 2.300, we constructed Plas-LuxI cell.<br />
Then we performed a reporter assay by using Lux reporter cell to characterize the function of Plas-LuxI. As the negative control of 3OC6HSL production, we prepared 3OC6HSL non-producer cell (ΔP-LuxI cell) that contains, in addition to Ptrc-LasR, promoterless-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008]) instead of Plas-LuxI (Fig2-1-3-1-5).<br />
[[File:positivefeedbackassay24tokyotech.png|550px|thumb|center|Fig2-1-3-1-5, Lux sender strain and Lux reporter strain]]<br />
<br />
<br />
The ΔP-LuxI cell does not produce 3OC6HSL even though 3OC12HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-6).<br />
[[File:positivefeedbackassay25tokyotech.png|500px|thumb|center|Fig2-1-3-1-6, How to perform 3OC12HSL-dependent 3OC6HSL production assay]]<br />
<br><br><br />
We prepared four conditions as follow.<br />
<br />
E) Culture containing Plas-LuxI cell without 3OC12HSL induction<br />
<br />
F) Culture containing Plas-LuxI cell with 3OC12HSL induction<br />
<br />
G) Culture containing ⊿P-LuxI cell without 3OC12HSL induction<br />
<br />
H) Culture containing ⊿P-LuxI cell with 3OC12HSL induction<br />
<br />
Using the supernatant of the four culture conditions, we performed the reporter assay. <br />
In the reporter assay, we used a Lux reporter strain that contains Ptet-LuxR and Plux-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395100 BBa_K395100]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.<br />
<br><br><br />
[[File:positivefeedbackassay26tokyotech.png|450px|thumb|right|Fig2-1-3-1-7, 3OC12HSL-dependent 3OC6HSL production]]<br />
<br />
Fig2-1-3-1-7 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) From this experiment, we confirmed that a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/C12#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br />
<br />
===Positive feedback assay ~Co-culture assay~===<br />
[[File:positivefeedbackassay17tokyotech.png|200px|thumb|right|Fig2-1-3-1-1, Positive feedback system in the cell-cell communication]]<br />
<br><br><br />
To accomplish complete positive feedback system, we mixed and co-cultured Plux-LasI cell and Plas-LuxI cell (Fig2-1-3-1-1). As a control co-culture, ⊿P-LasI cell and ⊿P-LuxI cell were mixed. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5nM) or 3OC12HSL (2.5nM) to the co-cultures. Including no-induction controls, we thus prepared six conditions (Fig2-1-3-1-8). To confirm that both 3OC6HSL and 3OC12HSL are produced in the positive feedback, the signals content in the supernatants of the co-cultures were evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-9).<br />
<br />
[[File:positivefeedbackassay27tokyotech.png|700px|thumb|center|Fig2-1-3-1-8, Six conditions prepared for positive feedback assay<br />
]]<br />
[[File:positivefeedbackassay28tokyotech.png|500px|thumb|center|Fig2-1-3-1-9, How to evaluate the positive feedback<br />
]]<br />
In these co-cultures, following behavior would be expected. In the condition I, 3OC6HSL induces 3OC12HSL production of Plux-LasI cell. Then, 3OC12HSL synthesized by Plux-LasI cell induces Plas-LuxI cell. The concentration of 3OC6HSL thus increases compared to the initial amount. Increased amount of 3OC6HSL induces higher production of 3OC12HSL. As a result, the production of both signals increased in the condition I. In this situation, the concentration of 3OC6HSL is higher than the initial concentration of 3OC6HSL (ideal positive feedback behavior). Similarly in the condition II, total amount of 3OC12HSL also increases compared to the initial amount. In the conditions III and VI, no signal is produced because of the lack of inducer. In conditions IV and V, little amount of 3OC6HSL and 3OC12HSL are remained without degradation, respectively.<br />
<br><br><br><br />
[[File:positivefeedbackassay31tokyotech.png|550px|thumb|right|Fig2-1-3-1-10, Positive feedback assay<br />
]]<br />
<br><br />
Fig2-1-3-1-10 shows that the fluorescence intensity of the Lux reporter cell in the condition I was higher than that in the condition IV. Similarly, the fluorescence intensity of the Las reporter in the condition II was higher than that in the condition V. <br />
From these results and circuit design described above, it is suggested that the appearance of positive feedback where cooperation of Plas-LuxI cell and Plux-LasI cell increase the production of 3OC6HSL and 3OC12HSL. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/positivefeedback1#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br><br><br />
<br><br><br><br />
<br />
===Positive feedback assay~Time-dependent change assay~===<br />
We confirmed the appearance of the positive feedback from the co-culture assay above. To further confirm the positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. <br />
To observe time-dependent change of the positive feedback, we prepared four conditions of co-culture : Plux-LasI cell and Plas-LuxI cell coexisted in condition i, ΔP-LasI cell and Plas-LuxI cell in condition ii, Plux-LasI cell and ΔP-LuxI cell in condition iii, and ΔP-LasI cell and ΔP-LuxI cell in condition iv. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5 nM) to each co-culture.<br />
<br />
<br><br />
[[File:positivefeedbackassay85tokyotech.png|700px|thumb|center|Fig2-1-3-1-11,Four conditions prepared for Time-dependent change assay]]<br />
<br><br />
To estimate the concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback, the signals content in the supernatants of the co-cultures were collected at the time of 0, 0.5, 1, 2, 4 hours. The concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback was evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-11). Fig2-1-3-1-11 also shows ideal signal production of the positive feedback in the cell-cell communication.<br />
<br><br />
[[File:positivefeedbackassay86tokyotech.png|700px|thumb|center|Fig2-1-3-1-12,Time-dependent change assay]]<br />
<br><br />
<br />
Fig2-1-3-1-12 shows the characterization of time-dependent signal increase in the positive feedback in the cell-cell communication. Solid lines represent the fluorescence intensities of Las reporters in the four conditions. Dotted lines represent the fluorescence intensities of Lux reporters in the four conditions.<br />
<br />
<br />
As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the fig shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.<br />
<br />
<br />
Fig2-1-3-1-12 also suggests that the advantage in signal production in the presence of both signal producers, Plux-LasI cell and Plas-LuxI cell. Only when Plux-LasI cell and Plas-LuxI cell were cultured together and allowed to communicate with one another, the fluorescence intensity of Las reporter and Lux reporter increased drastically. On the other hand, when either Plux-LasI cell or Plas-LuxI cell existed (condition ii and iii), drastic rearing in the GFP expression was not observed. Especially in the condition iii, GFP in the las reporter cell is expressed to some extent (about 2-folds lower than in condition i). This behavior shows that the production of 3OC12HSL in the condition iii cannot exceed the production of 3OC12HSL in the positive feedback state. This is because ΔP-LuxI cell cannot produce any 3OC6HSL to activate Plux-LasI cell in this condition, though initially added 3OC6HSL activated Plux-LasI cell and Plux-LasI cell produced 3OC12HSL to some extent. Moreover, it goes without saying that, the fluorescence intensity of both Las reporter and Lux reporter remained low level through observation in the absence of both signal producer cell. <br />
<br />
<br />
In addition, low concentration of 3OC6HSL was detected by the Lux reporter in the condition ii, iii & iv, in which 3OC6HSL is not produced. However, fluorescence intensity in these conditions decreased with time. Therefore, it is indicated that these GFP expressions were derived from initially added 3OC6HSL. <br />
<br />
<br />
From these overall results, we further confirmed the positive feedback in the cell-cell communication and characterized time-dependent change of the positive feedback. Only when the two types of <I>E.coli</I> were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increased as compared with other conditions.<br />
<br />
<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/positivefeedback2#Materials_.26_Methods Materials and Methods]]<br />
<br><br><br><br />
<br />
===Conclusion of positive feedback system===<br />
In this study, we designed and implemented a positive feedback system in the cell-cell communication that is composed of the Plux-LasI cell and the Plas-LuxI cell. <br />
<br />
<br />
First, we confirmed that the Plux-LasI cell synthesized enough concentration of 3OC12HSL to induce the Las reporter cell and the Plas-LuxI cell synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell. In the process of the implementation, we constructed two new Biobrick parts Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]), the new Biobrick parts that can be regulated by the induction of 3OC6HSL and 3OC12HSL, respectively. <br />
<br />
By co-culturing of these two types of <I>E.coli</I>, we then confirmed that higher concentration of a signal than initial conditions was detected through production of the other signal. These results indicated appearance of the positive feedback. <br />
<br />
To further confirm the positive feedback system, we also characterized the time-dependent change of this positive feedback in the cell-cell communication. The result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly verifies the appearance of the positive feedback.<br />
<br />
Only when the two types of <I>E.coli</I> were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increase as compared to other conditions.<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3-2.</div><br />
<br />
==Band detect system==<br />
===Introduction of band detect system===<br />
====the original band detect system====<br />
To achieve our goals, we need the 3OC6HSL-dependent band detect system.<br />
<br />
[[File:luxtethybrid20tokyotech.png|700px|thumb|center|Fig2-1-3-2-1, <br>(a)the band detect system constructed by Ron Weiss Lab. <br>(b)Our new band detect system]]<br />
<br />
In 2005, the first band detect system in synthetic biology was constructed by Ron Weiss Lab.([[#Reference|[1]]]<br />
) In that system (Fig2-1-3-2-1(a)), 3OC6HSL-LuxR complex activates the expression of LacIM1 repressor and CI repressor. Furthermore, CI repressor represses the expression of LacI. Then, both LacIM1 repressor and LacI repressor repress the expression of GFP. <br />
When the concentration of 3OC6HSL is high, the expression of LacIM1 is promoted strongly. As a result, the expression of GFP is repressed. When the concentration of 3OC6HSL is moderate, it results in the moderate level of CI and LacIM1. Because the repression efficiency of CI is sufficiently high, the LacI expression remains repressed. However, in this situation, the concentration of LacIM1, whose repression efficiency is low, is not sufficient to repress the GFP expression. Thus, GFP is expressed. When the concentration of 3OC6HSL is low, the expression of CI is only at low level. This enables the expression of LacI, then the expression of GFP is repressed again.<br />
In this way, the expression of GFP is activated by the band detect system under the particular range of the concentration of 3OC6HSL.<br />
<br />
In this project, we invented the new band detect system (Fig2-1-3-2-1(b)). Compared with the old one, our new band detect system has a merit, that is constructed with fewer components. By reducing components in cells, we can avoid the useless complexity and the inhibition of cell growth.<br />
We employed the lux/tet hybrid promoter in the system. In the following, we described the details of our system.<br />
<br />
====our new band detect system====<br />
The band detect system for cell-cell communication system is composed of lux/tet hybrid promoter,Plux-TetR,and Pon-LuxR. In contrast to the Weiss lab’s band detect system, which take advantage of difference in activity between wild-type repressor and its mutant, we used a hybrid promoter for the system. The expression of target gene, LasI, is regulated by both of LuxR-3OC6HSL complex and TetR through binding of them to lux/tet promoter.<br />
The expression of TetR from lux promoter is also regulated by LuxR-3OC6HSL (Fig2-1-3-2-2). Thus, concentration of 3OC6HSL affects both of lux/tet hybrid promoter and lux promoter since Pon-LuxR constitutively express LuxR. <br />
[[File:luxtethybrid12tokyotech.png|500px|thumb|center|Fig2-1-3-2-2]]<br />
When the concentration of 3OC6HSL is initial level, LuxR-3OC6HSL complex activates the expression of LasI. Though TetR is also expressed, its concentration is not enough to repress the expression of LasI.(Fig2-1-3-2-3)<br />
[[File:luxtethybrid1tokyotech.png|500px|thumb|center|Fig2-1-3-2-3]]<br />
As the concentration of 3OC6HSL increases to moderate level gradually by the positive feedback system, the expression of TetR and LasI also increases. In this situation, the concentration of TetR is still not enough to repress the expression of LasI.(Fig2-1-3-2-4)<br />
[[File:luxtethybrid2tokyotech.png|500px|thumb|center|Fig2-1-3-2-4]]<br />
<br />
When the concentration of 3OC6HSL is high level, the concentration of TetR reaches to the enough level to repress LasI expression(Fig2-1-3-2-5). For the implementation of the band detect system, lux/tet hybrid promoter is required. <br />
[[File:luxtethybrid3tokyotech.png|500px|thumb|center|Fig2-1-3-2-5]]<br />
The repression of production of 3OC12HSL results in arrest of the 3OC6HSL supply from Romeo cell. Then the LuxR-3OC6HSL complex cannot form and the expression of LasI is stopped.(Fig2-1-3-2-6)<br />
[[File:luxtethybrid4tokyotech.png|500px|thumb|center|Fig2-1-3-2-6]]<br />
<br />
===Result===<br />
[[File:luxtethybrid5tokyotech.png|450px|thumb|right|Fig2-1-3-2-7,lux/tet hybrid promoter assay]]<br />
For construction of the band detect system, we developed a new part lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]). The lux/tet hybrid promoter, which is composed of a LuxR operator and two TetR operators, activates the expression of the downstream gene only when LuxR-3OC6HSL complex exists and active TetR does not exist. To characterize the function of the lux/tet hybrid promoter, we constructed a part, Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) by inserting the promoter in front of a GFP coding sequence. By using the reporter cell that contains Plux/tet-GFP and constitutive LuxR and TetR generator (PlacIq-LuxR-Ptrc-TetR), we measured the fluorescence intensity of the reporter cell. In the measurement, we confirmed the GFP expression under the four different combinations of two inducers, 3OC6HSL and aTc (anhydrous tetracycline). In the absence of the both inducers, the culture with lux-tet hybrid promoter-gfp showed the background–fluorescence intensity generated by promoterless-rbs-gfp on pSB3K3. The presence of either 3OC6HSL or aTc alone had little effect on increasing the fluorescence intensity. In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers(Fig2-1-3-2-7). This result confirmed that the assembly of the LuxR operator and the two TetR operators integrated the inputs of 3OCH6HSL and aTc into the output of GFP transcription. [[https://2012.igem.org/Team:Tokyo_Tech/Experiment/banddetect#Materials_.26_Method Materials and Methods]]<br />
<br />
===Discussion===<br />
[[File:luxtethybrid31tokyotech.png|400px|thumb|right|Fig2-1-3-2-8]]<br />
[[File:luxtethybrid32tokyotech.png|400px|thumb|right|Fig2-1-3-2-9]]<br />
<br />
In this study, we improved lux/tet hybrid promoter parts by developing a new lux/tet hybrid promoter that is regulated by LuxR-3OC6HSL and TetR. Even though a former team had reported a lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K176078 BBa_K176078]), their assay did not contain the inducer combination:3OC6HSL(-) and aTc(+).Thus, data of lux/tet hybrid promoter in Biobrick Registry was not sufficient(Fig2-1-3-2-9). In contrast, all four combinations were confirmed in our new Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025])<br />
Therefore, our work is considered as the improvement of the lux/tet hybrid promoter.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br />
<br />
<br />
</div><br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3-3.</div><br />
<br />
==communication-inverter dependent suicide system==<br />
<br />
Introduction:<br />
<br />
In order to reproduce the suicides of Romeo & Juliet’s, the communication dependent inverter system that express lysis protein only when the signals do not exist is required.To realize the communication dependent inverter system, we constructed Plas-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) and Plux-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) that express LacI to repress the target gene only when LasR-3OC12HSL and LuxR-3OC6HSL exist respectively.<br />
<br />
<br />
'''3OC12HSL dependent'''<br />
<br />
construction<br />
<br />
We constructed a 3OC12HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) by ligating PlasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).<br />
<br />
'''3OC6HSL dependent'''<br />
<br />
construction<br />
<br />
We constructed a 3OC6HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) by ligating Plux ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).<br />
<br />
</div><br />
</div><br />
</div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=Modeling=<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by wet experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling#Model_development Detailed descriptions for Modeling development]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div class="whitebox"><br />
<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|500px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4, the story of “Romeo and Juliet”]]<br />
<br><br />
'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
<br />
'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
<br />
'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
<br />
'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
<br />
<br><br><br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==Result2: Validation of three subsystems’ function==<br />
<br />
In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
<br />
<br />
'''(1)Positive feedback system'''<br />
<br />
To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
<br />
<br />
[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increase. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
<br />
<br />
'''(2)Band detect system'''<br />
<br />
Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
<br />
[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.(Fig2-1-4-8)<br />
<br />
<br />
[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
<br><br><br><br><br><br><br><br />
On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration(Fig2-1-4-9). However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
<br />
<br><br><br><br><br><br><br />
<br />
In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
<br><br><br />
'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,<br>(a)the concentration of Juliet signals <br>(b)the concentration of lysis proteins in Romeo cells <br>(c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
<br><br><br><br><br />
'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gene) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-11, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
<br><br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==a==<br />
a<br />
</div></div></div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<div></div><br />
5.</div><br />
<br />
=Application=<br />
In this project, we constructed the new cell-cell communication system. The special features of our system are as follows.<br />
<br />
1. One kind of signal activates the production of another one via the positive feedback system.<br />
<br />
2. The band detect system regulates the excessive production of LasI enzymes.<br />
<br />
3. Communication-inverter dependent suicide system controls the cell population.<br />
<br />
By applying these features, we would achieve a new system for manufacturing. <br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
<br />
(1) Sustainable manufacturing</div><br />
[[File:Application5tokyotech.png|400px|thumb|right|Fig2-1-5-1, the circuit with positive feedback system and band detect system]]<br />
<br />
Simulation shows that the collaboration of positive feedback system and band detect system (Fig2-1-5-1) produces the signals at the moderate level (the yellow line and the purple line in Fig2-1-5-2). <br />
[[File:Application85tokyotech.png|800px|thumb|center|Fig2-1-5-2, the time-dependent change of 3OC12HSL concentration in four situations]]<br />
<br />
Fig2-1-5-2 shows the time-dependent change of 3OC12HSL concentration in the culture. In the collaboration of positive feedback system and band detect system, the concentration of 3OC12HSL settles into a moderate steady state (the yellow line or the purple line), compared with high concentration in the simple positive feedback system (the red line). When two types of E.coli are cultured separately, 3OC12HSL is not produced (the blue line). <br />
In addition, by changing parameters, we can control the concentration in the final steady state (the yellow line and the purple line).<br />
<br />
We propose inserting the coding region of desired proteins to downstream region of LuxI or LasI in this system. In that case, the production of desired proteins is activated only when two types of E.coli are co-cultured. Then, once it starts, the increase of production is accelerated by the positive feedback system. Furthermore, the band detect system plays an important role. The band detect system regulates the excessive production of proteins in the host cells. Thereby, it is expected that the host cells can avoid growth inhibition caused by metabolic imbalance.<br />
<br />
As described above, by applying this system, we would lighten the metabolic burdens on the host cells and achieve sustainable manufacturing system. Moreover, it is suggested that we can control the concentration in the final steady state.<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;"><br />
(2) Division of labor</div><br />
It is known that microbial consortia can perform even more complicated tasks through the division of labor than individual strains. For example, when the products require multiple steps to convert the substrates, the system with multiple strains which is dedicated to each step has two advantages, compared to the system with a single strain. First, by limiting the number of exogenous elements in the host cells, metabolic imbalance in the cells reduces. Second, it is possible to improve the reaction efficiency by isolating the engineered circuits dedicated to each reaction. <br />
<br />
In our system, the two types of E.coli communicate with each other with signal molecules and control the expression of proteins mutually. By applying this system, we would achieve more efficient manufacturing, for example, the simultaneous conversion of sugar mixtures at similar rates.<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
6.</div><br />
<br />
=Reference=<br />
<br />
1. Basu S, Gerchman Y, Collins CH, Arnold FH, & Weiss R (2005) A synthetic multicellular system for programmed pattern formation. Nature 434(7037):1130-1134.<br />
<br />
2. You L, Cox RS, Weiss R, & Arnold FH (2004) Programmed population control by cell-cell communication and regulated killing. Nature 428:868-871.<br />
<br />
3. Balagadde FK, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4:187<br />
<br />
4. J Biol Eng. (2008) co-fermentation strategy to consume sugar mixtures effectively Published online 2008 February 27. <br />
<br />
5. Brenner K, You L, Arnold FH Engineering microbial consortia: a new frontier in synthetic biology Trends in Biotechnology [2008, 26(9):483-489]</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ModelingTeam:Tokyo Tech/Modeling2012-10-27T03:15:28Z<p>Nakayama: /* a */</p>
<hr />
<div>{{tokyotechcss}}<br />
{{tokyotechmenubar}}<br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
__TOC__<br />
<br />
=Modeling=<br />
<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#Model_development Detailed descriptions for Modeling]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div class="whitebox"><br />
<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|300px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4: the story of “Romeo and Juliet”]]<br />
<br><br><br><br><br />
'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
<br />
'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
<br />
'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
<br />
'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
<br />
<br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==Result2: Validation of three subsystems’ function==<br />
<br />
In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
<br />
<br />
'''(1)Positive feedback system'''<br />
<br />
To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
<br />
<br />
[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increases. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
<br />
<br />
'''(2)Band detect system'''<br />
<br />
Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
<br />
[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.<br />
<br />
<br />
[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
<br><br><br><br><br><br><br><br />
On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration. However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
<br />
<br><br><br><br><br><br><br />
<br />
In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
<br />
'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,(a)the concentration of Juliet signals (b)the concentration of lysis proteins in Romeo cells (c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
<br />
<br />
'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gone) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells population somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in fig4-9, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
<br><br><br><br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==a==<br />
a<br />
</div></div></div></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ModelingTeam:Tokyo Tech/Modeling2012-10-27T03:14:42Z<p>Nakayama: /* Result2: Validation of three subsystems’ function */</p>
<hr />
<div>{{tokyotechcss}}<br />
{{tokyotechmenubar}}<br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
__TOC__<br />
<br />
=Modeling=<br />
<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#Model_development Detailed descriptions for Modeling]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div class="whitebox"><br />
<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|300px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4: the story of “Romeo and Juliet”]]<br />
<br><br><br><br><br />
'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
<br />
'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
<br />
'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
<br />
'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
<br />
<br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==Result2: Validation of three subsystems’ function==<br />
<br />
In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
<br />
<br />
'''(1)Positive feedback system'''<br />
<br />
To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
<br />
<br />
[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increases. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
<br />
<br />
'''(2)Band detect system'''<br />
<br />
Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
<br />
[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.<br />
<br />
<br />
[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
<br><br><br><br><br><br><br><br />
On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration. However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
<br />
<br><br><br><br><br><br><br />
<br />
In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
<br />
'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,(a)the concentration of Juliet signals (b)the concentration of lysis proteins in Romeo cells (c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
<br />
<br />
'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gone) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells population somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in fig4-9, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
<br><br><br><br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==a==<br />
a<br />
</div></div></div><br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ModelingTeam:Tokyo Tech/Modeling2012-10-27T03:13:29Z<p>Nakayama: /* Result2: Validation of three subsystems’ function */</p>
<hr />
<div>{{tokyotechcss}}<br />
{{tokyotechmenubar}}<br />
<br><br><br />
<br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
__TOC__<br />
<br />
=Modeling=<br />
<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#Model_development Detailed descriptions for Modeling]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div class="whitebox"><br />
<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|300px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4: the story of “Romeo and Juliet”]]<br />
<br><br><br><br><br />
'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
<br />
'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
<br />
'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
<br />
'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
<br />
<br><br><br><br />
</div><br />
<div class="whitebox"><br />
<br />
==Result2: Validation of three subsystems’ function==<br />
<br />
In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
<br />
<br />
'''(1)Positive feedback system'''<br />
<br />
To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
<br />
<br />
[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increases. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
<br />
<br />
'''(2)Band detect system'''<br />
<br />
Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
<br />
[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.<br />
<br />
<br />
[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
<br><br><br><br><br><br><br><br />
On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration. However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
<br />
<br><br><br><br><br><br><br />
<br />
In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
<br />
'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,(a)the concentration of Juliet signals (b)the concentration of lysis proteins in Romeo cells (c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
<br />
<br />
'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gone) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells population somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in fig4-9, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
<br><br><br><br><br><br><br />
</div></div><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
<div class="whitebox"><br />
<br />
==a==<br />
a<br />
</div></div></div><br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/ModelingTeam:Tokyo Tech/Modeling2012-10-27T03:12:02Z<p>Nakayama: /* Result2: Validation of three subsystems’ function */</p>
<hr />
<div>{{tokyotechcss}}<br />
{{tokyotechmenubar}}<br />
<br><br><br />
<br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 30px;"><br />
__TOC__<br />
<br />
=Modeling=<br />
<br />
To build our cell-cell communication system, we have constructed and characterized several important parts and subsystems by experiments. However, it is unconfirmed whether <I>E.coli</I> can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.<br />
<br />
==Model development==<br />
To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in Fig2-1-4-1. The Variables are described in Table2-1-4-1.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#Model_development Detailed descriptions for Modeling]]<br />
<br />
[[File:tokyotechModeling1.png|350px|thumb|left|Fig2-1-4-1,The equations used in the model]]<br />
[[File:tokyotechModeling20.png|350px|thumb|left|Table2-1-4-1, the variables]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div class="whitebox"><br />
<br />
==Result1: Whether our circuit can reproduce “Romeo and Juliet”==<br />
To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.<br />
<br />
[[File:tokyotechModelingresult1.png|300px|thumb|center|Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.]]<br />
We set the initial values of variables and the parameters as follows: [[https://2012.igem.org/Team:Tokyo_Tech/Project/modeling2#the_initial_values_of_variables_and_the_parameters see more]]<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig2-1-4-3, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig2-1-4-4: the story of “Romeo and Juliet”]]<br />
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'''In the yellow area of Fig2-1-4-3''', the concentration of two signals increases. It looks as if Romeo and Juliet fall in love. <br />
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'''In the green area of Fig2-1-4-3''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep. <br />
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'''In the blue area of Fig2-1-4-3''', lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself. <br />
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'''In the pink area of Fig2-1-4-3''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.<br />
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==Result2: Validation of three subsystems’ function==<br />
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In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function.<br />
We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in Juliet cells under the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.<br />
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'''(1)Positive feedback system'''<br />
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To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback.<br />
In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.<br />
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[[File:tokyotechMode6ing1.png|200px|thumb|left|Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system]]<br />
[[File:tokyotechMode7ing1.png|200px|thumb|left|Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system]]<br />
[[File:tokyotechMode8ing1.png|200px|thumb|left|Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system]]<br />
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In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increases. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.<br />
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'''(2)Band detect system'''<br />
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Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals. <br />
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[[File:tokyotechMode9ing1.png|250px|thumb|right|Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals]]<br />
In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.<br />
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[[File:tokyotechMode10ing1.png|250px|thumb|right|Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo signals]]<br />
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On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration. However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.<br />
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In this manner, the validity of Band detect system in Juliet cells was proven by modeling.<br />
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'''(3)Communication-inverter dependent suicide system – in Romeo cell'''<br />
[[File:tokyotechMode11ing1.png|250px|thumb|right|Fig2-1-4-10,(a)the concentration of Juliet signals (b)the concentration of lysis proteins in Romeo cells (c)the number of Romeo cells]]<br />
To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.<br />
<br />
On the Line(1) in Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). <br />
On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.<br />
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'''(4)Communication-inverter dependent suicide system – in Juliet cell'''<br />
[[File:tokyotechMode12ing1.png|250px|thumb|right|Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells]]<br />
Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.<br />
<br />
On the Line(1) in Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gone) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells population somewhat increases (Fig2-1-4-11(c)). <br />
On the other hand, on the Line(2) in fig4-9, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).<br />
<br />
In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.<br />
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==a==<br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"></div>Nakayamahttp://2012.igem.org/Team:Tokyo_TechTeam:Tokyo Tech2012-10-27T03:10:07Z<p>Nakayama: /* Ⅰ-2 Positive Feedback of cell-cell communication */</p>
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HOME</div><br />
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__NOTOC__<br />
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=Project overview=<br />
[[File:tokyotechprojectoverview.png|250px|right]]<br />
A love story contains several processes. Two people fall in love and their love burning wildly. However, no forever exists in the world, in most occasions, love will eventually burn to only a pile of ashes of the last remaining wind drift away. In our project, we have recreated the story of "Romeo and Juliet" by Shakespeare vividly by two kinds of <I>Escherichia coli</I> [[#Ⅰ cell-cell communication|cell-cell communication]]. We aim to generate a circuit involving regulatory mechanism of positive feedback rather than commonly-used negative feedback to control the fate of <I>E.coli</I> by signaling between two types of <I>E.coli</I>. Besides, Rose represents love. We are the first iGEM group ever to synthesize [[#.E2.85.A1__P.283HB.29_Production|P(3HB) ]] (a kind of bio-plastics) by using our new biobrick part, representing rose .<br />
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=Ⅰ cell-cell communication=<br />
"Romeo and Juliet" is the drama by dramatist William Shakespeare of England. The stage is city Verona, Italy in the 14th century. Romeo met Juliet. Two people fell in love instantly. In this project, we will recreate the love story of "Romeo and Juliet".<br />
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[[https://2012.igem.org/Team:Tokyo_Tech/Project Detailed descriptions for Cell-cell communication]]<br />
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==Ⅰ-1 story==<br />
We make our cute <I>E.coli</I> play “Romeo and Juliet” which is one of Shakespeare’s most famous plays. In this project, we define the signal that <I>E.coli</I> produce as the romantic feeling of Romeo and Juliet. In this project, we will recreate the love story of "Romeo and Juliet", by using "Cell-cell communication"<br />
The story that we reproduce is divided into four scenes.<br />
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Click on the Scene.<br />
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[[File:tokyotechhstory1.png|300px|left]] <br />
<p>'''(Scene 1)''' Romeo meets and falls in love with Juliet. Once the love between two people stimulates each other, they become deeply attached and cannot live without each other.<br><br><br>First, to represent the condition that their love stimulates each other in “Scene1 Fall in love”, we designed a positive feedback system in which the production of a signal activates the production of the other signal. [[https://2012.igem.org/Team:Tokyo_Tech/Project#Assays_for_Positive_feedback_system Detailed descriptions for Positive feedback system]]<br />
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<p>'''(Scene 2)''' However, Juliet knows that their love will not be accepted by society because of family feud. To keep their relationship, Juliet plans to pretend to be dead. She takes a sleeping potion that makes her fall into a deathlike sleep. <br><br><br>Second, we applied a 3OC6HSL-dependent band detect system to represent “Scene2 Juliet’s deathlike sleep” by the stop of 3OC12HSL production in Juliet cell. When the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR is enough level to repress the expression of LasI, As a result, the production of 3OC12HSL is stopped though cell Juliet is alive. [[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Detailed descriptions for Band detect system]]<br />
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[[File:tokyotechhstory3.png|300px|left]] <br />
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<p>'''(Scene 3)''' Romeo has heard of Juliet’s death without knowing the fact that Juliet is alive. Romeo decides to commit suicide by taking poison in response to Juliet’s “deathlike sleep”. <br><br><br>Third, to realize “Scene3 Romeo’s suicide” after Juliet fell into the deathlike sleep, we designed communication-inverter dependent suicide system in Romeo cell. When Juliet cell is in deathlike sleep, supply of 3OC12HSL is stopped though Juliet cell is alive. In the absence of 3OC12HSL, the lysis gene is expressed and Romeo cell dies.[[https://2012.igem.org/Team:Tokyo_Tech/Project#communication-inverter_dependent_suicide_system Detailed descriptions for communication-inverter dependent suicide system]]<br />
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[[File:tokyotechhstory4.png|300px|left]] <br />
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<p>'''(Scene 4)''' Juliet awakes to find Romeo dead beside her. She decides to commit suicide in response to Romeo’s suicide. She stabs herself with a dirk.<br><br><br>Finally, to realize “Scene4 Juliet’s suicide” in response to Romeo’s suicide, we also designed communication-inverter dependent suicide system in Juliet cell. After the death of Romeo cell, supply of 3OC6HSL is stopped, and the expression of LacI is stopped. As a result, the lysis gene is expressed and Juliet cell dies.[[https://2012.igem.org/Team:Tokyo_Tech/Project#communication-inverter_dependent_suicide_system Detailed descriptions for communication-inverter dependent suicide system]]<br />
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==Ⅰ-2 Positive Feedback of cell-cell communication==<br />
[[File:positivefeedbackassay20tokyotech.png|800px|thumb|center|Fig1-2,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay.7ETime-dependent_change_assay.7E Time-dependent change assay]]]]<br />
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To accomplish the positive feedback system in the cell-cell communication, we designed and constructed two types of genetically engineered <I>E.coli</I>, 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) and 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell). <br />
<br>For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5 nM) to the co-cultures. Only when the two types of signal producer cells were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increase as compared to other conditions. <br>As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the fig shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.<br />
<br>In the process of the implementation, we also confirmed two modules, the 3OC6HSL-dependent 3OC12HSL production module and the 3OC12HSL-dependent 3OC6HSL production module, which constituted our positive feedback system. In these module, two new Biobrick parts, Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]), were characterized their functions.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Project Detailed descriptions for Cell-cell communication]]<br />
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Other experiments for basis of "Romeo & Juliet"<br />
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[[File:positivefeedbackassay18tokyotech.png|170px|thumb|left|Fig1-3,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC6HSL-dependent_3OC12HSL_production_module Go to <br>"Construction_of<br>the_3OC6HSL-dependent<br>3OC12HSL_production"]]]]<br />
[[File:positivefeedbackassay19tokyotech.png|170px|thumb|left|Fig1-4,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC12HSL-dependent_3OC6HSL_production_module Go to <br>"Construction_of<br>the_3OC12HSL-dependent<br>3OC6HSL_production"]]]]<br />
[[File:positivefeedbackassay30tokyotech.png|150px|thumb|left|Fig1-5,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Positive_feedback_assay_.7ECo-culture_assay.7E Go to <br>"Co-culture assay"]]]]<br />
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[[File:positivefeedbackassay80tokyotech.png|150px|thumb|left|Fig1-6,[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to <br>"Band detect system"]]]]<br />
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==Ⅰ-3 Modeling==<br />
[[File:tokyotechModelingresult2.png|250px|thumb|right|Fig1-7, time-dependent change of the concentrations of the two signals.]]<br />
[[File:tokyotechStory.png|200px|thumb|right|Fig1-8, the story of “Romeo and Juliet”]]<br />
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'''Result: Whether our circuit can reproduce “Romeo and Juliet”'''<br />
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To confirm the feasibility of the cell-cell communication system, we developed an ordinary differential equation model and simulated the system under typical experimental conditions. Fig1-7 shows the result of the simulation. As described below, the behavior of the signal concentration is consistent with the development of the “Romeo and Juliet” story.<br />
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'''In the yellow area of Fig1-7''', the concentration of two signals increases. It represents Scene1.<br />
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'''In the green area of Fig1-7''', as the concentration of Romeo signals increases to some extent, the concentration of Juliet signals starts to decline. It represents Scene2. <br />
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'''In the blue area of Fig1-7''', lysis gene is expressed in Romeo cell in response to decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. It represents Scene3.<br />
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'''In the pink area of Fig1-7''', lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. It represents Scene4.<br />
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[[https://2012.igem.org/Team:Tokyo_Tech/Project#Modeling Detailed descriptions for Modeling]]<br />
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=Ⅱ P(3HB) Production=<br />
==Ⅱ-1 Story==<br />
[[File:tokyotech PHA make rose.png|300px|thumb|right|fig1-9,Rose silhouette on the LB agar plate containing Nile red.]]<br />
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There is what of the famous scene of "Romeo and Juliet"<br />
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JULIET: O Romeo, Romeo! why are you Romeo? Deny your father and refuse your name;</div><br />
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ROMEO: Shall I hear more, or shall I speak at this?</div><br />
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JULIET: It's but your name that is my enemy; you are yourself, though not a Montague. O, be some other name! What's in a name? that which we call a rose By any other name would smell as sweet.</div><br />
we will recreate the rose come out in the lines of the famous drama "Romeo and Juliet"<br />
by the synthesis of P(3HB).<br />
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==Ⅱ-2 P(3HB) production==<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroixyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs.<br />
In our project, we also draw rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm Detailed descriptions for P(3HB) production]]<br />
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=Human Practice=<br />
[[File:tokyotech human7.png|200px|right]]<br />
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Taku Nakayama, and Mai Miura (members of Tokyo_Tech iGEM team) have participated in a science cafe as assistants for the event. The two members supported people who are not specialist in biology to plan an imaginary synthetic biology project, to be evaluated from public points of view, and to upgrade the project in accordance with the evaluations.<br />
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[[https://2012.igem.org/Team:Tokyo_Tech/HumanPractice.htm Detailed descriptions for Human practice] <br />
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<br></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T03:06:46Z<p>Nakayama: /* Application */</p>
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesizes the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so <I>E.coli</I> synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29. Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
<br />
<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:15:55Z<p>Nakayama: /* 4-4 Optimization of the best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesizes the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so <I>E.coli</I> synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:15:18Z<p>Nakayama: /* 4-4 Optimization of the best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br><br><br><br><br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is the rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesizes the polymer in maximum content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so <I>E.coli</I> synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:07:26Z<p>Nakayama: /* Application */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br><br><br><br><br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain a lot of carbon sources, so we think that the rate-limiting step is carbon sources in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon sources,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have stronger water-repellent by increasing real surface area. From literature data, contact angle of P(3HB) sheets is about 100°.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:03:23Z<p>Nakayama: /* 4-4 Optimization of the best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br><br><br><br><br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain a lot of carbon sources, so we think that the rate-limiting step is carbon sources in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon sources,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:02:53Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization of the best culture condition to synthesize P(3HB)==<br />
<br />
To figure out best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br><br><br><br><br />
<br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain a lot of carbon sources, so we think that the rate-limiting step is carbon sources in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon sources,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T02:01:39Z<p>Nakayama: /* 4-3 Confirmation of P(3HB) by GC/MS */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
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<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
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The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
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The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
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3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
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==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
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The culture result is shown in Fig. 2-2-4-4-4.<br />
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[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
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*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain a lot of carbon sources, so we think that the rate-limiting step is carbon sources in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon sources,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
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[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-27T01:01:29Z<p>Nakayama: /* D. Optimization best culture condition to synthesize P(3HB) */</p>
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<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A. Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B. Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C. Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==D. Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|550px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
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<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
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<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
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=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/File:Tokyotech_PHB9.pngFile:Tokyotech PHB9.png2012-10-27T00:58:19Z<p>Nakayama: uploaded a new version of &quot;File:Tokyotech PHB9.png&quot;</p>
<hr />
<div></div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T00:28:01Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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<div id="tokyotech" style=" font:bold ;left ; font-size: 50px; color: #1E90FF; padding: 10px;"><br />
P(3HB) Production </div><br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as carbon sources.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain a lot of carbon sources, so we think that the rate-limiting step is carbon sources in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon sources,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T00:21:05Z<p>Nakayama: /* Achivement */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimised best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon source,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T00:18:18Z<p>Nakayama: /* What is PHAs? */</p>
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P(3HB) Production </div><br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
<br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon source,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T00:09:36Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon source,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.(the comparison of condition 7 & 8 and 9&10)<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-27T00:07:17Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough carbon source,so we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:51:16Z<p>Nakayama: /* D Optimization best culture condition to synthesize P(3HB) */</p>
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{{tokyotechmenubar}}<br />
<br><br><br />
<br />
<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==D Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:50:40Z<p>Nakayama: /* Construction of pha-C1-A-B1 in Biobrick format */</p>
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<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:50:17Z<p>Nakayama: /* D Optimization best culture condition to synthesize P(3HB) */</p>
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<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:48:44Z<p>Nakayama: /* D Examine best culture condition to synthesize P(3HB) */</p>
<hr />
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<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 conditions for 48h. This culture is done in test tubes. Composition of LB and TB medium is shown in Fig2-2-4-5-1.<br />
<br />
<br />
[[File:tokyotech PHB2.png|350px|thumb|center|Fig2-2-4-5-1, Composition of LB & TB]]<br />
<br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-5-2, Structure of Pantothenic acid<br />
]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA. The culture result is shown in Fig2-2-4-5-3.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-3, Culture results of ten conditions]]<br />
<br />
Both in LB and TB, when we added glucose and PA-Ca, culturing at 37°C, we got maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
<br />
In addition, according to condition 4 & 5, PA-Ca is not used as a carbon source. Since <I>E.coli</I> has rich carbon source in TB medium, according to condition 7 & 8 and 9 & 10, the glycolytic pathway may become a rate-limiting step, so polymer production would be increased by adding PA-Ca.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#E_Preparation_for_GC.2FMS Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
<br />
2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
<br />
</div><br />
<br />
2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:48:02Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:47:34Z<p>Nakayama: /* C Confirmation of P(3HB) by GC/MS */</p>
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<div class="whitebox"><br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D Examine best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 conditions for 48h. This culture is done in test tubes. Composition of LB and TB medium is shown in Fig2-2-4-5-1.<br />
<br />
<br />
[[File:tokyotech PHB2.png|350px|thumb|center|Fig2-2-4-5-1, Composition of LB & TB]]<br />
<br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-5-2, Structure of Pantothenic acid<br />
]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA. The culture result is shown in Fig2-2-4-5-3.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-3, Culture results of ten conditions]]<br />
<br />
Both in LB and TB, when we added glucose and PA-Ca, culturing at 37°C, we got maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
<br />
In addition, according to condition 4 & 5, PA-Ca is not used as a carbon source. Since <I>E.coli</I> has rich carbon source in TB medium, according to condition 7 & 8 and 9 & 10, the glycolytic pathway may become a rate-limiting step, so polymer production would be increased by adding PA-Ca.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#E_Preparation_for_GC.2FMS Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br />
<br><br />
<br />
=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
<br />
2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
<br />
2.4 42° C,30secs, heatshock<br />
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2.5 Add 160ul of SOC into the tube<br />
<br />
2.6 Incubate the the cells at 37° C for 30mins<br />
<br />
2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
</div><br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
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1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
<br />
1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
<br />
1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
<br />
<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
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2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
<br />
2.1.2 Add some culture solution into each tube.<br />
<br />
2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
<br />
2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
<br />
<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
<br />
2.2.3 Freeze dry for 3 days.<br />
<br />
</div><br />
2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
<br />
2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
<br />
2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
<br />
</div><br />
==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
<br />
3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
<br />
7. Set in GC/MS.<br />
<br />
==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
1 Preparing<br />
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<br />
1.1 2x LB solution (autoclaved) 100ml<br />
<br />
Tryptone 2g<br />
<br />
Yeast extract 1g<br />
<br />
NaCl 2g<br />
<br />
<br />
1.2 2x TB solution (autoclaved) 100ml<br />
<br />
Tryptone 2.4g<br />
<br />
Yeast extract 4.8g<br />
<br />
Glycerol 1.6ml<br />
<br />
K2HPO4 1.88g<br />
<br />
KH2PO4 0.44g<br />
<br />
<br />
1.3 50% glucose (autoclaved) 100ml<br />
<br />
Glucose 50g<br />
<br />
Pure water up to 100ml<br />
<br />
<br />
1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
<br />
Pantothenic acid Ca 9.53g<br />
<br />
Pure water up to 20ml<br />
<br />
</div><br />
2 Polymer producing media<br />
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<br />
2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
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<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
<br />
</div><br />
<br />
2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
<br />
<br />
2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:47:16Z<p>Nakayama: /* 4-3 Confirmation of P(3HB) by GC/MS */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Experiment/PHBTeam:Tokyo Tech/Experiment/PHB2012-10-26T23:46:37Z<p>Nakayama: /* C Confirmation of P(3HB) by GC/MS */</p>
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=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
==A Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<br />
==B Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
==C Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==D Examine best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 conditions for 48h. This culture is done in test tubes. Composition of LB and TB medium is shown in Fig2-2-4-5-1.<br />
<br />
<br />
[[File:tokyotech PHB2.png|350px|thumb|center|Fig2-2-4-5-1, Composition of LB & TB]]<br />
<br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-5-2, Structure of Pantothenic acid<br />
]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA. The culture result is shown in Fig2-2-4-5-3.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-3, Culture results of ten conditions]]<br />
<br />
Both in LB and TB, when we added glucose and PA-Ca, culturing at 37°C, we got maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
<br />
In addition, according to condition 4 & 5, PA-Ca is not used as a carbon source. Since <I>E.coli</I> has rich carbon source in TB medium, according to condition 7 & 8 and 9 & 10, the glycolytic pathway may become a rate-limiting step, so polymer production would be increased by adding PA-Ca.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#E_Preparation_for_GC.2FMS Protocol]]<br />
<br />
=Construction of pha-C1-A-B1 in Biobrick format=<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]<br />
To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]<br />
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=Protocol=<br />
<br><br />
<br />
==A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV==<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
1 Preparation of LB agar medium plate containing Nile red and Glucose<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C<br />
<br />
1.2 After the autoclave, add Chloramphenicol(final 25ug/ml), Nile red and glucose(final 20g/L) to the LB agar solution when it cools down.<br />
<br />
1.3 Make LB agar medium plates with the mixture.<br />
<br />
<br />
</div><br />
2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Thaw the competent cells JM109 at 4° C<br />
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2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.<br />
<br />
2.3 Put the tube into ice for 15mins<br />
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2.4 42° C,30secs, heatshock<br />
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2.5 Add 160ul of SOC into the tube<br />
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2.6 Incubate the the cells at 37° C for 30mins<br />
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2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.<br />
<br />
2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.<br />
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_P.283HB.29_synthesized_on_colonies Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]]<br />
<br />
==B.P(3HB) production in cells and preparation before the confirmation with Nile blue A==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
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1 Production of PHB<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
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1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop<br />
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1.2 Culture the colony in LB solution for 16hrs at 37 ° C <br />
<br />
1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.<br />
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1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.<br />
<br />
1.5 Set all flasks into autoclave<br />
<br />
1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.<br />
<br />
1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.<br />
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<br />
<br />
[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]<br />
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2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1 Collection of PHBs in JM109<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.1.1 Weigh empty 50ml falcon tube without lid and make a record.<br />
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2.1.2 Add some culture solution into each tube.<br />
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2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.<br />
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2.1.4 4 ° C, 5000G, 10mins in centrifuge.<br />
<br />
2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.<br />
<br />
2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.<br />
<br />
2.1.7 Remove the supernatant and add a little amount of water<br />
<br />
2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.<br />
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<br />
</div><br />
2.2 Freeze drying (lyophilization)<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2.2.1 Poke several holes on the tubes’ parafilm with toothpick.<br />
<br />
2.2.2 Set the tubes on the freeze drying machine.<br />
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2.2.3 Freeze dry for 3 days.<br />
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2.3 Stain PHB accumulated dried cells with Nile blue A before observation<br />
<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
2.3.1 Acquire dried cells after freeze drying<br />
<br />
2.3.2 Put a small amount of cells on the slide glass<br />
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2.3.3 Add water on the cells and heat the slide glass immobilize the cells<br />
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2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes <br />
<br />
2.3.5 Wash excess Nile blue A with 8% acetic acid solution<br />
</div><br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_P.283HB.29_accumulated_in_cells Back to "4-2 Confirmation of P(3HB) accumulated in cells"]]<br />
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==C. Preparation for GC/MS==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-3_Confirmation_of_P.283HB.29_by_GC.2FMS Back to "4-3 Confirmation of P(3HB) by GC/MS"]]<br />
<br />
<br />
1. Put 10mg of dried cells in glass tubes.<br />
<br />
2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.<br />
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3. Incubate tubes at 100℃ for 140min.<br />
<br />
4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.<br />
<br />
5. Remove the organic layer, filtered.<br />
<br />
6. Add internal standard fluid to organic layer.<br />
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7. Set in GC/MS.<br />
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==D. Optimization best condition to synthesize P(3HB)==<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
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<br />
1 Preparing<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
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1.1 2x LB solution (autoclaved) 100ml<br />
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Tryptone 2g<br />
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Yeast extract 1g<br />
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NaCl 2g<br />
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1.2 2x TB solution (autoclaved) 100ml<br />
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Tryptone 2.4g<br />
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Yeast extract 4.8g<br />
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Glycerol 1.6ml<br />
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K2HPO4 1.88g<br />
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KH2PO4 0.44g<br />
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1.3 50% glucose (autoclaved) 100ml<br />
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Glucose 50g<br />
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Pure water up to 100ml<br />
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1.4 1M Pantothenic acid Ca (Filter sterilized)<br />
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Pantothenic acid Ca 9.53g<br />
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Pure water up to 20ml<br />
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2 Polymer producing media<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
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2.1 LB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
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2x LB 5ml<br />
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50% Glc 400ul<br />
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1M Pantothenic acid Ca 200ul<br />
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Cm(25mg/ml) 12ul<br />
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Pure water(autoclaved) 4.388ml<br />
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</div><br />
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2.2 TB, 2% Glc, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
1M Pantothenic acid Ca 200ul<br />
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Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.388ml<br />
</div><br />
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2.3 LB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
<br />
2.4 TB, 2% Glc, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
50% Glc 400ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.588ml<br />
</div><br />
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2.5 LB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.6 TB, 20mM Pantothenic acid Ca, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Pantothenic acid Ca 200ul<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.788ml<br />
<br />
</div><br />
2.7 LB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x LB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
2.8 TB, 30μg/ml Chloramphenicol, 10ml<br />
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;"><br />
<br />
2x TB 5ml<br />
<br />
Cm(25mg/ml) 12ul<br />
<br />
Pure water(autoclaved) 4.988ml<br />
<br />
</div><br />
</div><br />
<br />
3. Culture and collection<br />
<br />
3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.<br />
<br />
3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.<br />
<br />
3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.<br />
<br />
3.4 Remove supernatant and suspend with pure water.<br />
<br />
3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.<br />
<br />
3.6 Freeze in -20℃.<br />
<br />
3.7 Freeze-dry for 72 hrs.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-4_Optimization_best_culture_condition_to_synthesize_P.283HB.29 Back to "Optimization best culture condition to synthesize P(3HB)"]]<br />
<br />
<br />
</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:40:12Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
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5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:39:53Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br />
<br />
<br />
The culture result is shown in Fig. 2-2-4-4-4.<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:36:14Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|700px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:36:03Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|700px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br><br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:35:17Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|700px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
<br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:34:45Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|600px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
<br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:34:28Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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{{tokyotechtop}}<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 50px; color: #1E90FF; padding: 10px;"><br />
P(3HB) Production </div><br />
</div class="whitebox"><br />
<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|600px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br />
<br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
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<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:33:31Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|600px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br><br><br><br><br />
<br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
[[File:tokyotech PHB2.png|400px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:28:52Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|480px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:25:58Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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<div id="tokyotech" style=" font:bold ;left ; font-size: 50px; color: #1E90FF; padding: 10px;"><br />
P(3HB) Production </div><br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|480px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:24:39Z<p>Nakayama: /* Application */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|480px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
<br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:24:01Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|480px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
*“Dried cells (g/L)” are amount of the cells in the medium after culturing.<br />
<br />
*“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
*“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:22:24Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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<div class="whitebox"><br />
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|480px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
“Dried cells (g/L)” are amount of cells in the medium after culturing.<br />
<br />
“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T23:22:03Z<p>Nakayama: /* 4-4 Optimization best culture condition to synthesize P(3HB) */</p>
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P(3HB) Production </div><br />
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[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|300px|thumb|right|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
<br><br><br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br><br><br />
<br><br><br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
“Dried cells (g/L)” are amount of cells in the medium after culturing.<br />
<br />
“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB2#Protocol Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T19:19:01Z<p>Nakayama: /* Achivement */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
==4-2 Confirmation of P(3HB) accumulated in cells==<br />
<br />
To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
<br />
[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
]]<br />
<br />
<br />
<br />
==4-3 Confirmation of P(3HB) by GC/MS==<br />
<br />
We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
<br />
[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Examine_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
<br />
[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
<br />
==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
<br />
To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
<br />
[[File:tokyotech PHB9.png|800px|thumb|center|Fig2-2-4-4-1, different conditions]]<br />
<br />
[[File:tokyotech PHB2.png|350px|thumb|center|Fig2-2-4-4-2, Composition of LB & TB]]<br />
<br />
[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
]]<br />
<br><br><br />
Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
<br><br><br><br><br><br />
<br />
<br />
[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
<br />
“Dried cells (g/L)” are amount of cells in the medium after culturing.<br />
<br />
“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
<br />
“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
<br />
The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#E_Preparation_for_GC.2FMS Protocol]]<br />
<br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
5.</div><br />
<br />
=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D_Polymer_extraction_and_purification Protocol]]<br />
<br />
<br />
[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
<br />
<br />
<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
<br><br />
6.</div><br />
<br />
=Reference=<br />
<br />
[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
<br />
[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
<br />
[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
<br />
[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
<br />
[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
<br />
[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
<br />
[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayamahttp://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htmTeam:Tokyo Tech/Projects/PHAs/index.htm2012-10-26T19:18:36Z<p>Nakayama: /* Achivement */</p>
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P(3HB) Production </div><br />
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;"> <br />
[[File:tokyotech PHA make rose.png|400px|thumb|right|Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.]]<br />
__TOC__<br />
<br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
1.</div><br />
=Achivement=<br />
We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(PHAs). This is the first Biobrick part to synthesize P(3HB), a kind of PHAs. We identified the products as P(3HB) by GC/ MS, and optimise best culture condition to synthesize P(3HB).<br />
In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).<br />
<br><br><br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
2.</div><br />
<br />
=What is PHAs?=<br />
<br />
Polyhydroxyalkanoates(PHAs) are biological polyester synthesized by a wide range of bacteria, and can be produced by fermentation from renewable carbon sources such as sugars and vegetable oils. These polyesters are biodegradable thermoplastics and elastomers, which exhibit interesting material properties. PHAs are also a kind of bio plastics, which can be biodegraded a lot faster than fossil-fuel plastics in the environment. Poly-3-hydroxybutyrate, P(3HB) is the most common type of PHAs. P(3HB) is synthesized by the enzymes coded in the gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.<br />
[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of PHA synthesis (<I>pha C1-A-B1</I>) from <I>Ralstonia eutropha</I> H16.]]<br />
<br><br><br />
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.<br />
<br />
<br />
<br />
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)<br />
<br />
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)<br />
<br />
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)<br />
<br><br><br><br><br><br />
[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]<br />
<br />
<br />
The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutropha</I> H16 is shown in Fig2-2-2-2. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in PhaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in PhaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).([[#Reference|[1][2]]]<br />
)<br />
<br />
<br><br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
3.</div><br />
<br />
=Construction of <I>phaC1-A-B1</I> in Biobrick format=<br />
In this study, we constructed a part containing <I>phaC1-A-B1</I> in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#Construction_of_pha-C1-A-B1_in_Biobrick_format Construction of <I>PHA</I>-C1-A-B1 in Biobrick format]]<br />
This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_trial_of_PHAs_by_past_teams Production trial of <I>PHA</I>s by past teams]]<br />
<br><br><br><br><br><br><br><br />
<br />
<br />
<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;"><br />
4.</div><br />
<br />
=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=<br />
<br />
To synthesize P(3HB) by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>pha C1-A-B1</I> part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([[#Reference|[5]]]<br />
). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.<br />
<br />
<br />
<br />
==4-1 Confirmation of P(3HB) synthesized on colonies==<br />
<br />
We observed the accumulation of P(3HB) in the <I>E.coli</I> colonies on Nile red positive medium under UV. Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of PHAs in the colonies can be directly monitored([[#Reference|[3][4][5]]]<br />
). We cultured the transformant on LB agar medium plates with Nile red. After several days, colonies storing P(3HB) were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored P(3HB).<br />
Fig2-2-4-1-1 is the photographs of <I>E.coli</I> colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1-1A show that the accumulated P(3HB) in cells was stained by Nile red. This result indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). Fig2-2-4-1-1B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. Fig2-2-4-1-2 shows the difference between cells storing P(3HB) and those not storing P(3HB) on one plate. The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing P(3HB), we drew a rose silhouette on the LB agar plate containing Nile red (Fig2-2-4-1-3).[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#A_.P.283HB.29_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]<br />
[[File:tokyotech PHA Nilered1.png|300px|thumb|left|Fig2-2-4-1-1 <br>Fig2-2-4-1-1A: <I>E.coli</I> JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation <br />
<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]<br />
<br />
[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB). <br>Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation. <br>Green rectangle: with PlasI-gfp gene, no PHB accumulation]]<br />
[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]<br />
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==4-2 Confirmation of P(3HB) accumulated in cells==<br />
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To confirm the accumulation condition of P(3HB) in <I>E.coli</I> with a microscope, we stained the P(3HB) with Nile blue A reagent. Nile blue A is also used to detect the existence of P(3HB) and has no toxicity to the cells([[#Reference|[5]]]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. <br />
Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>pha C1-A-B1</I> gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in Fig2-2-4-2-1A are the accumulated P(3HB) in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized P(3HB). In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#B.P.283HB.29_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]<br />
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[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|<br />
Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A <br />
Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A<br />
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==4-3 Confirmation of P(3HB) by GC/MS==<br />
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We successfully identified the products by K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products by GC/ MS, the products are methylated because 3HB is difficult to measure. Fig. 2-2-4-3-1 shows the GC/ MS result of the products by K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that <I>E.coli</I> synthesized P(3HB) correctly.<br />
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[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Examine_best_condition_to_synthesize_P.283HB.29 Protocol]]<br />
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]<br />
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==4-4 Optimization best culture condition to synthesize P(3HB)==<br />
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To examine best culture condition, we tried culturing <I>E.coli</I> JM109 in 10 different conditions for 48h. Each condition is shown in Fig.2-2-4-1. Composition of LB and TB medium is shown in Fig. 2-2-4-4-2.<br />
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[[File:tokyotech PHB9.png|600px|thumb|center|Fig2-2-4-4-1, different conditions]]<br />
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[[File:tokyotech PHB2.png|350px|thumb|center|Fig2-2-4-4-2, Composition of LB & TB]]<br />
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[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid<br />
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Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.<br />
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[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]<br />
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“Dried cells (g/L)” are amount of cells in the medium after culturing.<br />
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“Polymer content rate (%)” is rate of the polymer in the dried cells.<br />
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“Polymer concentration (g/L)” is amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.<br />
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The results show that TB medium is much better than LB medium to synthesize P(3HB).<br />
Both in LB and TB, in the 37°C culturing containing glucose and PA-Ca, <I>E.coli</I> synthesized maximum polymer content rate. However, the growth of <I>E.coli</I> in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, <I>E.coli</I> synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so <I>E.coli</I> may use them as a carbon source.<br />
In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as a carbon source. LB medium doesn’t contain much carbon source, so we think that the rate-limiting step is carbon source in LB. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contain enough we think that the rate-limiting step may be the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#E_Preparation_for_GC.2FMS Protocol]]<br />
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5.</div><br />
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=Application=<br />
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]<br />
The achievement of our project “P(3HB) Production” is that we registered available P(3HB) synthetic gene in Biobrick parts. We can control the expression of the P(3HB) synthetic gene spatially by using combination of Biobrick parts. What we want to claim as an example of the spatial manipulation of gene expression is water-repellent. A stronger water-repellent requires hydrophobicity as well as the increase in real surface area that can be achieved as ruggedness of P(3HB) adsorbed on particular surface. If we can control the expression of the P(3HB) synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available.<br />
We made P(3HB) sheets. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension.When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data.[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D_Polymer_extraction_and_purification Protocol]]<br />
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[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]<br />
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6.</div><br />
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=Reference=<br />
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[1] Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650<br />
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[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.<br />
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[3] Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836<br />
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[4] Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol 2. issue 4.e-00109-11<br />
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[5] Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80<br />
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[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912<br />
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[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)</div>Nakayama