Team:Tokyo Tech/Experiment/PHB

From 2012.igem.org

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P(3HB) Production </div>
 
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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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__TOC__
 
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<br>
 
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
 
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1.</div>
 
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=Achivement=
 
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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.
 
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In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of  P(3HB).
 
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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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<div class="whitebox">
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=PHB production by <I>E.coli</I> & Confirmation of PHB=
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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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To synthesize PHB by <I>E.coli</I>, we transformed <I>E.coli</I> JM109 with the constructed <I>PHA</I>C1-A-B1 part 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([[#Reference|[5]]]
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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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). As a negative control, we transformed <I>E.coli</I> JM109 with PlasI-gfp on pSB1C3.
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<br><br>
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Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.
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==4-1 Confirmation of PHB synthesized on colonies==
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We observed the accumulation of PHB 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 <I>PHA</I>-accumulating and non-accumulating colonies. Nile red in the agar medium doesn’t affect the growth of the cells, and the accumulation of <I>PHA</I>s 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 PHB were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored PHB.
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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 PHB 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 PHB. 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 PHB and those not storing PHB on one plate. The cells in blue rectangle area are the cells with PHB synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing PHB, 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/Projects/PHAs/detail/index.htm#A_.PHB_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 PHB accumulation]]
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The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
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[[File:tokyotech PHA Nilered3.png|300px|thumb|left|Fig2-2-4-1-2, Difference between cells storing PHB and cells not storing PHB. <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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<br><br><br><br><br><br>
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The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA  reductase (PhaB)
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==Confirmation of PHB accumulated in cells==
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The C gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)
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To confirm the accumulation condition of PHB in <I>E.coli</I> with a microscope, we stained the PHB with Nile blue A reagent. Nile blue A is also used to detect the existence of PHB 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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<br><br><br><br><br>
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Fig2-2-4-2-1 is the photograph of dried <I>E.coli</I> (with <I>PHA</I>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 PHB in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized PHB. In the photograph of negative control (Fig2-2-4-2-1B), no remarkable fluorescent area was observed.[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#B.PHB_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]
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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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[[File:tokyotech PHA Nileblue1.png|500px|thumb|center|
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Fig2-2-4-2-1A, <I>E.coli</I> JM109 dried cells with PHB accumulation stained by Nile blue A
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Fig2-2-4-2-1B, <I>E.coli</I> JM109 dried cells without PHB accumulation stained by Nile blue A
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]]
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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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=Construction of pha-C1-A-B1 in Biobrick format=
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)
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[[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"]]
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[[File:tokyotech PHA biobrick.png|350px|thumb|right|Fig1,construction of phaC1-A-B1]]
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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]).
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[[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"]]
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<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;">
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=Protocol=
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3.</div>
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<br>
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=Construction of <I>phaC1-A-B1</I> in Biobrick format=
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===A .PHB production on colonies and preparation before confirmation with Nile red under UV===
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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]).[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#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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<br><br><br><br><br><br><br>
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_PHB_synthesized_on_colonies Go to the project page  "4-1 Confirmation of PHB synthesized on colonies"]]
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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1 Preparation of LB agar medium plate containing Nile red and Glucose
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4.</div>
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<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C
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=P(3HB) production by <I>E.coli</I> & Confirmation of P(3HB)=
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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.
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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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1.3 Make LB agar medium plates with the mixture.
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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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</div>
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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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2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109
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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/Projects/PHAs/detail/index.htm#A_.PHB_production_on_colonies_and_preparation_before_confirmation_with_Nile_red_under_UV Protocol]]
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<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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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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2.1 Thaw the competent cells JM109 at 4° C
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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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2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.
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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>
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==4-2 Confirmation of P(3HB) accumulated in cells==
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2.3 Put the tube into ice for 15mins
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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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2.4 42° C,30secs, heatshock
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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/Projects/PHAs/detail/index.htm#B.PHB_production_in_cells_and_preparation_before_the_confirmation_with_Nile_blue_A Protocol]]
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[[File:tokyotech PHA Nileblue1.png|500px|thumb|center|
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2.5 Add 160ul of SOC into the tube
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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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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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2.6 Incubate the the cells at 37° C for 30mins
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5.</div>
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=Possible Synbio research area by using our achievement=
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2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.
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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 “PHAs Production” is that we registered available PHA synthetic gene in Biobrick parts.
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2.8    Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.
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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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</div>
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<div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #0000FF; padding: 2px;">
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<br>
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-1_Confirmation_of_PHB_synthesized_on_colonies Go to the project page  "4-1 Confirmation of PHB synthesized on colonies"]]
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6.</div>
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===B.PHB production in cells and preparation before the confirmation with Nile blue A===
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_PHB_accumulated_in_cells Go to the project page  "4-2 Confirmation of PHB accumulated in cells"]]
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1 Production of PHB
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<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop
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1.2 Culture the colony in LB solution for 16hrs at 37 ° C
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1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.
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1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.
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1.5 Set all flasks into autoclave
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1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.
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1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.
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[[File:tokyotech PHA 7.png|250px|thumb|center|Fig2. air permeable lids]]
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</div>
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2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope
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<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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2.1 Collection of PHBs in JM109
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<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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2.1.1 Weigh empty 50ml falcon tube without lid and make a record.
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2.1.2 Add some culture solution into each tube.
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2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.
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2.1.4 4 ° C, 5000G, 10mins in centrifuge.
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2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.
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2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.
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2.1.7 Remove the supernatant and add a little amount of water
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2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.
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</div>
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2.2 Freeze drying (lyophilization)
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<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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2.2.1 Poke several holes on the tubes’ parafilm with toothpick.
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2.2.2 Set the tubes on the freeze drying machine.
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2.2.3 Freeze dry for 3 days.
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=Reference=
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</div>
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2.3 Stain PHB accumulated dried cells with Nile blue A before observation
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<div id="tokyotechprotocol2" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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2.3.1 Acquire dried cells after freeze drying
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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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2.3.2 Put a small amount of cells on the slide glass
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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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2.3.3 Add water on the cells and heat the slide glass immobilize the cells
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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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2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes
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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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2.3.5 Wash excess Nile blue A with 8% acetic acid solution
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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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</div>
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</div>
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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)
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#4-2_Confirmation_of_PHB_accumulated_in_cells Go to the project page  "4-2 Confirmation of PHB accumulated in cells"]]

Revision as of 06:37, 21 October 2012

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Contents

PHB production by E.coli & Confirmation of PHB

To synthesize PHB by E.coli, we transformed E.coli JM109 with the constructed PHAC1-A-B1 part on pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). E.coli JM109 is used to synthesize PHB, because it tends to have a high density accumulation of PHB([5] ). As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.

4-1 Confirmation of PHB synthesized on colonies

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 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 PHB were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored PHB. Fig2-2-4-1 is the photographs of E.coli colonies on Nile red positive medium taken under UV. The orange colonies in Fig2-2-4-1A show that the accumulated PHB 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 PHB. 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 PHB and those not storing PHB on one plate. The cells in blue rectangle area are the cells with PHB synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control. Using the cells storing PHB, 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 PHB accumulation
Fig2-2-4-1-2, Difference between cells storing PHB and cells not storing PHB.
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
























Confirmation of PHB accumulated in cells

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 PHB 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 PHAC1-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 PHB in the cells. This result also indicates that part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001] synthesized PHB. 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 PHB accumulation stained by Nile blue A Fig2-2-4-2-1B, E.coli JM109 dried cells without PHB accumulation stained by Nile blue A

Construction of pha-C1-A-B1 in Biobrick format

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Fig1,construction of phaC1-A-B1

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]).

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Protocol


A .PHB production on colonies and preparation before confirmation with Nile red under UV

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1 Preparation of LB agar medium plate containing Nile red and Glucose

1.1 Autoclave a LB agar(final 40g/L) solution at 120 ° C

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.

1.3 Make LB agar medium plates with the mixture.


2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109

2.1 Thaw the competent cells JM109 at 4° C

2.2 Add the target DNA 3ul into 1.5ml tube, then add in 50ul the thawed competent cells.

2.3 Put the tube into ice for 15mins

2.4 42° C,30secs, heatshock

2.5 Add 160ul of SOC into the tube

2.6 Incubate the the cells at 37° C for 30mins

2.7 Spread the resulting culture on LB agar medium plate with a large cone rod.

2.8 Incubate the plate at 37° C for 36hrs then cells the plate into 4° C room for 2-3 days.


[Go to the project page "4-1 Confirmation of PHB synthesized on colonies"]

B.PHB production in cells and preparation before the confirmation with Nile blue A

[Go to the project page "4-2 Confirmation of PHB accumulated in cells"]

1 Production of PHB

1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop

1.2 Culture the colony in LB solution for 16hrs at 37 ° C

1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.

1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.

1.5 Set all flasks into autoclave

1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.

1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.


Fig2. air permeable lids

2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope

2.1 Collection of PHBs in JM109

2.1.1 Weigh empty 50ml falcon tube without lid and make a record.

2.1.2 Add some culture solution into each tube.

2.1.3 Set the tubes into centrifuge and make sure that the label faces outside.

2.1.4 4 ° C, 5000G, 10mins in centrifuge.

2.1.5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.

2.1.6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.

2.1.7 Remove the supernatant and add a little amount of water

2.1.8 Cover the tubes with double layers of parafilms and fully freeze them.


2.2 Freeze drying (lyophilization)

2.2.1 Poke several holes on the tubes’ parafilm with toothpick.

2.2.2 Set the tubes on the freeze drying machine.

2.2.3 Freeze dry for 3 days.

2.3 Stain PHB accumulated dried cells with Nile blue A before observation

2.3.1 Acquire dried cells after freeze drying

2.3.2 Put a small amount of cells on the slide glass

2.3.3 Add water on the cells and heat the slide glass immobilize the cells

2.3.4 Stain the cells with 1% Nile blue A solution (water) for 8 minutes

2.3.5 Wash excess Nile blue A with 8% acetic acid solution


[Go to the project page "4-2 Confirmation of PHB accumulated in cells"]