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

From 2012.igem.org

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<I>PHA</I>s Production </div>
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<I>PHB</I> Production </div>
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1.</div>
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=Achivement=
=Achivement=
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We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(<I>PHA</I>s). This is the first Biobrick part to synthesize <I>PHA</I>s.
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We made a new biobrick part and succeeded in synthesizing Polyhydroxyalkanoates(<I>PHB</I>). This is the first Biobrick part to synthesize <I>PHB</I>.
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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 <I>PHA</I>s.
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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 <I>PHB</I>.
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=What is <I>PHA</I>?=
=What is <I>PHA</I>?=
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Polyhydroxyalkanoates(<I>PHA</I>s) 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. <I>PHA</I>s 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 <I>PHA</I>s. P(3HB) is synthesized by the enzymes coded in the gene of <I>PHA</I> synthesis (<I>PHA</I>C1-A-B1) from <I>Ralstonia eutropha</I> H16.
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Polyhydroxyalkanoates(<I>PHB</I>) 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. <I>PHB</I> 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 <I>PHB</I>. P(3HB) is synthesized by the enzymes coded in the gene of <I>PHB</I>ynthesis (<I>PHA</I>C1-A-B1) from <I>Ralstonia eutropha</I> H16.
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[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of <I>PHA</I> synthesis (<I>PHA</I>C1-A-B1) from <I>Ralstonia eutropha</I> H16.]]
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[[File:tokyotech PHA whatsPHA.png|300px|thumb|left|Fig2-2-2-1, Gene of <I>PHB</I>ynthesis (<I>PHA</I>C1-A-B1) from <I>Ralstonia eutropha</I> H16.]]
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Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.
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The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA  reductase (<I>PHA</I>B)
The B gene encodes for the 246 amino acids protein, acetoacetyl-CoA  reductase (<I>PHA</I>B)
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The C gene encodes for the 589 amino acids protein, <I>PHA</I> Synthase (<I>PHA</I>C)
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The C gene encodes for the 589 amino acids protein, <I>PHB</I>ynthase (<I>PHA</I>C)
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[[File:tokyotech PHA whatsPHA2.png|150px|thumb|left|Fig2-2-2-2, synthesis mechanism of P(3HB)]]
[[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 Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutro<I>PHA</I></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 <I>PHA</I>A). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in <I>PHA</I>B). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of <I>PHA</I> synthase (<I>PHA</I>C).([[#Reference|[1][2]]]
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The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in <I>Ralstonia eutro<I>PHA</I></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 <I>PHA</I>A). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in <I>PHA</I>B). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of <I>PHB</I>ynthase (<I>PHA</I>C).([[#Reference|[1][2]]]
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=Construction of <I>PHA</I>C1-A-B1 in Biobrick format=
=Construction of <I>PHA</I>C1-A-B1 in Biobrick format=
In this study, we constructed a part containing <I>PHA</I>C1-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]]
In this study, we constructed a part containing <I>PHA</I>C1-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 <I>PHA</I>s 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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This is the first Biobrick part which worked as expected though some teams had tried to synthesize <I>PHB</I> 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>PHB</I> by past teams]]
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==4-1 Confirmation of PHB synthesized on colonies==
==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 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>PHB</I> in the colonies can be directly monitored([[#Reference|[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.
). 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 <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]]
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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=Possible Synbio research area by using our achievement=
=Possible Synbio research area by using our achievement=
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, <I>PHA</I> gene expression spatially manipulated]]
[[File:tokyotech PHA perspective.png|200px|thumb|right|Fig2-2-5-1, <I>PHA</I> gene expression spatially manipulated]]
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The achievement of our project “<I>PHA</I>s Production” is that we registered available <I>PHA</I> synthetic gene in Biobrick parts.
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The achievement of our project “<I>PHB</I> Production” is that we registered available <I>PHB</I>ynthetic gene in Biobrick parts.
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We can control the expression of the <I>PHA</I> 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 <I>PHA</I> adsorbed on particular surface. If we can control the expression of the <I>PHA</I> 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.
+
We can control the expression of the <I>PHB</I>ynthetic 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 <I>PHA</I> adsorbed on particular surface. If we can control the expression of the <I>PHB</I>ynthetic 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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[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
[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 (<I>PHA</I>) homeostasis: the role of the <I>PHA</I> synthase, 2003, Nat. Prod. Rep.,20, 445–457.
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[2] Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (<I>PHA</I>) homeostasis: the role of the <I>PHB</I>ynthase, 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
[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

Revision as of 03:05, 21 October 2012

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PHB 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(PHB). This is the first Biobrick part to synthesize PHB. In our project, we also drew rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of PHB.

2.

What is PHA?

Polyhydroxyalkanoates(PHB) 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. PHB 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 PHB. P(3HB) is synthesized by the enzymes coded in the gene of PHBynthesis (PHAC1-A-B1) from Ralstonia eutropha H16.

Fig2-2-2-1, Gene of PHBynthesis (PHAC1-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, PHBynthase (PHAC)




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


The pathway and regulation of Poly[(R)-3-hydroxybutyrate], P(3HB) synthesis in Ralstonia eutro<I>PHA</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 PHBynthase (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(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 PHB in the past iGEM.[Production trial of PHB by past teams]







4.

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 (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 PHB 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 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 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 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 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 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
5.

Possible Synbio research area by using our achievement

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

The achievement of our project “PHB Production” is that we registered available PHBynthetic gene in Biobrick parts. We can control the expression of the PHBynthetic 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 PHBynthetic 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.


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 PHBynthase, 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