Team:Tokyo Tech/Projects/PHAs/index.htm

From 2012.igem.org

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PHAs Production </div>
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P(3HB) Production </div>
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[[File:tokyotech PHA make rose.png|300px|thumb|right|fig1,]]
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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.]]
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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1.</div>
=Achivement=
=Achivement=
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We made a new biobrick part and succeeded synthesizing Polyhydroixyalkanoates(PHAs).
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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).
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In our project, we designed rose silhouette to enhance the balcony scene of “Romeo and Juliet” by the synthesis of PHAs.
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=What is PHA?=
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In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).
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<br><br>
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Polyhydroixyalkanoates(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 (phaC1-A-B1) from Ralstonia eutropha H16.
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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2.</div>
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=What is PHAs?=
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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.
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[[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.]]
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<br><br>
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.
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[[File:tokyotech PHA whatsPHA.png|250px|thumb|left|fig1]]
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[[File:tokyotech PHA whatsPHA2.png|150px|thumb|right|fig2]]
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The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
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The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)
The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)
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[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]
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The pathway and regulation of P(3HB) synthesis in Ralstonia eutropha H16 is shown in Fig2. Acetyl CoA is metabolized from glucose by glycolysis and Pyruvate dehydrogenase complex (PDC). 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) polymers are then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC). In most of the cases, PHA synthase determines the characteristic of PHA being synthesized in microorganism.
 
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=Other teams=
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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]]]
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)
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In this study, we constructed a part containing pha-C1-A-B1 in Biobrick format.
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<br>
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We introduce some attempts in the past iGEM to show how great our work is in iGEM. There is no Biobrick part which worked as expected though some teams had tried to synthesize PHA in the past.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Other_Teams see more]]
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=Construction of phaC1-A-B1 in Biobrick format=
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To make pha-C1-A-B1 operon meets Biobrick format, we used artificial composition of gene from IDT/MBL. Wild type pha-C1-A-B1 operon contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. In this chemically synthesized DNA, coding is optimized for E.coli. 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]).
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3.</div>
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Construction_of_pha-C1-A-B1_in_Biobrick_format see more]]
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=PHB production by <I>E.coli</I> & Confirmation of PHB=
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=Construction of <I>phaC1-A-B1</I> in Biobrick format=
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To synthesize PHB by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed phaC1-A-B1 parts on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). <I>E.coli</I> JM109 is used to synthesize PHB, because it tends to have a high density accumulation of PHB.As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Production_of_PHAs_by_E.coli see more]]
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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]]
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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]]
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[[File:tokyotech PHA Nilered1.png|200px|thumb|right|fig6,E.coli JM109 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]. , PHB accumulation) ]]
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[[File:tokyotech PHA Nilered2.png|150px|thumb|right|fig6,E.coli JM109 (plasI-gfp, no PHB accumulation)]]
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()  
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4.</div>
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We observed the accumulation of PHB in the E.coli 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 produces a strong orange fluorescence (emission maximum,598nm) with an excitation wavelength of 543nm (maximum) upon binding to PHA-granules in cells of R.eutropha (Degelau et al.1995) H16. Nile red in the agar medium doesn’t affect the growth of the cells, and the occurrence of PHAs in the colonies can be directly monitored.
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This method is quite sensitive and results in fluorescent colonies of PHA-positive strains. So we cultured the transformant on LB agar medium plates with 0.5μg/ml Nile Red and 2% glucose at 37°C for  30 hours, then we transferred the plates to 4°C room. After 115 hours, colonies with PHB would be stained Red by Nile red when observed under UV. This showed that the transformant had stored PHB.FIG1 is the photograph of E.coli(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 fucntioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.  
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=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=
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[[File:tokyotech PHA Nileblue1.png|200px|thumb|right|fig6,E.coli JM109 (BBa_K934001, PHB accumulation)]]
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[[File:tokyotech PHA Nileblue2.png|150px|thumb|right|fig6 E.coli JM109 (plasI-gfp, no PHB accumulation)]]
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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]]]
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). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.
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==4-1 Confirmation of P(3HB) synthesized on colonies==
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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]]]
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). 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).
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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]]
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[[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
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<br>Fig2-2-4-1-1B: <I>E.coli</I> JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation]]
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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]]
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[[File:tokyotech PHA make rose.png|150px|thumb|right|Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.]]
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<br><br><br><br><br><br>
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==4-2 Confirmation of P(3HB) accumulated in cells==
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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.
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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]]
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[[File:tokyotech PHA Nileblue1.png|800px|thumb|center|
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Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with P(3HB) accumulation stained by Nile blue A
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Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without P(3HB) accumulation stained by Nile blue A
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]]
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==4-3 Confirmation of P(3HB) by GC/MS==
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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.
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[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#C._Preparation_for_GC.2FMS Protocol]]
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[[File:tokyotech PHB1.png|800px|thumb|center|Fig2-2-4-3-1, Result of GC/MS]]
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==4-4 Optimization of the best culture condition to synthesize P(3HB)==
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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.
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[[File:tokyotech PHB9.png|500px|thumb|left|Fig2-2-4-4-1, different conditions]]
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<br><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.
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[[File:tokyotech PHB2.png|380px|thumb|left|Fig2-2-4-4-2, Composition of LB & TB]]
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<br><br>
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[[File:tokyotech PHB3.png|400px|thumb|right|Fig2-2-4-4-3, Structure of Pantothenic acid
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]]
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<br><br><br><br>
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(ⅱ)Nile Blue A
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To confirm the accumulation condition of PHB in E.coli with a microscope, we stained the PHB with Nile blue A reagent.Nile blue A is also used to detect the existence of PHBs and has no toxicity to the cells.We carried out the observation after the procedures stated below. First,We cultured a small amount of the transformant (3ml) in LB medium for 17 hrs. Then,we added the cultured trandformant(1%) into LB medium(with 2% glucose) and shaking cultured it under aerobic condition with an air permeable lid for 96 hrs. After the cultivation we recovered the bacteria with PHA accumulation by centrifugation. We were able to get the dried cells after the procedure of lyophilization. Before taking the photographs,we stained the dried cells with 1% Nile blue A solution for 8 minutes and washed the extra Nile blue with 8% acetic acid solution afterwards. We then took photographs of the sample under fluorescence microscope. FIG1 is the photograph of E.coli(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 functioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.
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=Perspective=
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[[File:tokyotech PHA perspective.png|200px|thumb|right|fig6,]]
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The achievement of our project “Organic synthesis of PHA” is that we registered available PHA synthetic gene in Biobrick parts.
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We can control the expression of the PHA 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 PHA adsorbed on particular surface. If we can control the expression of the PHA synthetic gene spatially by using genetic parts which are registered in Biobrick parts, the application of a super water-repellent sheet will become available. We note this as to the future prospects of our project.
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The culture result is shown in Fig. 2-2-4-4-4.
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[[File:tokyotech PHB4.png|800px|thumb|center|Fig2-2-4-5-4, Culture results of ten conditions]]
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*“Dried cells (g/L)” is the amount of the cells in the medium after culturing.
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*“Polymer content rate (%)” is the rate of the polymer in the dried cells.
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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”.
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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)
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[[https://2012.igem.org/Team:Tokyo_Tech/Experiment/PHB#D._Optimization_of_the_best_culture_condition_to_synthesize_P.283HB.29. Protocol]]
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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5.</div>
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=Application=
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[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, PHA synthesis gene expression spatially manipulated]]
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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.
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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]]
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[[File:tokyotech PHB6.png|600px|thumb|center|Fig2-2-5-2, P(3HB) sheet]]
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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6.</div>
=Reference=
=Reference=
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Text
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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
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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.
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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
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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
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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
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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
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[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol(2006),  24:1257-62

Latest revision as of 03:56, 27 October 2012

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P(3HB) Production
Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.

Contents


1.

Achivement

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).

In our project, we also drew rose silhouette to reproduce the balcony scene of “Romeo and Juliet” by the synthesis of P(3HB).

2.

What is PHAs?

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 (pha C1-A-B1) from Ralstonia eutropha H16.

Fig2-2-2-1, Gene of PHA synthesis (pha C1-A-B1) from Ralstonia eutropha H16.



Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.


The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)

The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)

The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)




Fig2-2-2-2, synthesis mechanism of P(3HB)


The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB), synthesis in Ralstonia eutropha 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).([1][2] )


3.

Construction of phaC1-A-B1 in Biobrick format

In this study, we constructed a part containing phaC1-A-B1 in Biobrick format([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]).[Construction of PHA-C1-A-B1 in Biobrick format] This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[Production trial of PHAs by past teams]







4.

P(3HB) production by E.coli & Confirmation of P(3HB)

To synthesize P(3HB) by E.coli, we transformed E.coli JM109 with the constructed pha C1-A-B1 part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). E.coli JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB)([5] ). As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.


4-1 Confirmation of P(3HB) synthesized on colonies

We observed the accumulation of P(3HB) in the E.coli 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([3][4][5] ). 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). Fig2-2-4-1-1 is the photographs of E.coli 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).[Protocol]

Fig2-2-4-1-1
Fig2-2-4-1-1A: E.coli JM109 colonies with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation
Fig2-2-4-1-1B: E.coli JM109 colonies with PlasI-gfp gene, no P(3HB) accumulation
Fig2-2-4-1-2, Difference between cells storing P(3HB) and cells not storing P(3HB).
Blue rectangle: with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] gene, PHB accumulation.
Green rectangle: with PlasI-gfp gene, no PHB accumulation
Fig2-2-4-1-3, Rose silhouette on the LB agar plate containing Nile red.
























4-2 Confirmation of P(3HB) accumulated in cells

To confirm the accumulation condition of P(3HB) in E.coli 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([5]). Before the observation, we stained the dried cells with Nile blue A solution. We then took photographs of the sample under fluorescence microscope. Fig2-2-4-2-1 is the photograph of dried E.coli (with pha C1-A-B1 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.[Protocol]

Fig2-2-4-2-1A, E.coli JM109 dried cells with P(3HB) accumulation stained by Nile blue A Fig2-2-4-2-1B, E.coli JM109 dried cells without P(3HB) accumulation stained by Nile blue A


4-3 Confirmation of P(3HB) by GC/MS

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 E.coli synthesized P(3HB) correctly. [Protocol]

Fig2-2-4-3-1, Result of GC/MS


4-4 Optimization of the best culture condition to synthesize P(3HB)

To figure out best culture condition, we tried culturing E.coli 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.

Fig2-2-4-4-1, different conditions



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.


Fig2-2-4-4-2, Composition of LB & TB



Fig2-2-4-4-3, Structure of Pantothenic acid













The culture result is shown in Fig. 2-2-4-4-4.

Fig2-2-4-5-4, Culture results of ten conditions
  • “Dried cells (g/L)” is the amount of the cells in the medium after culturing.
  • “Polymer content rate (%)” is the rate of the polymer in the dried cells.
  • “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”.


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, E.coli synthesized the polymer in maximum content rate. However, the growth of E.coli 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, E.coli synthesized polymer (condition 9 & 10). We think that TB medium had glycerol and a lot of yeast extra, and then E.coli 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 E.coli 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) [Protocol]


5.

Application

Fig2-2-5-1, PHA synthesis gene expression spatially manipulated

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.


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°.[Protocol]


Fig2-2-5-2, P(3HB) sheet




6.

Reference

[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

[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat. Prod. Rep.,20, 445–457.

[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

[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

[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

[6] Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry Chemical Physics(2009), vol 11.897-912

[7] Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol(2006), 24:1257-62