PHAs Production

Contents 1 Achivement 2 What is PHA? 3 Construction of phaC1-A-B1 in Biobrick format 4 PHB production by E.coli & Confirmation of PHB 4.1 (i)Confirmation of PHB synthesized on colonies 4.2 (ii)Confirmation of PHB accumulated in cells 5 Perspective 6 Reference

Achivement

We made a new biobrick part and succeeded synthesizing Polyhydroixyalkanoates(PHAs). In our project, we designed rose silhouette to enhance the balcony scene of “Romeo and Juliet” by the synthesis of PHAs.

What is PHA?

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.

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)

The pathway and regulation of Poly[(R)-3-hydroxybutyrate] ,P(3HB) synthesis in Ralstonia eutropha H16 is shown in Fig2. 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] )

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]).[See more] This is the first Biobrick part which worked as expected though some teams had tried to synthesize PHAs in the past iGEM.[See more]

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 parts 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.

(i)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. ([4][5] ). We cultured the transformant on LB agar medium plates with Nile red. After 4 days, colonies storing PHB were stained orange by Nile red when observed under UV. This result indicates that transformant synthesized and stored PHB. FIG1 is the photographs of E.coli colonies on Nile Red positive medium taken under UV. The orange colonies in FIG1.a show that the accumulated PHB in cells was stained by Nile red. This result indicates that part BBa_K934001 synthesized PHB. FIG1.b is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies. FIG2 shows the difference between cells storing PHB and those not storing PHB. 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. (fig4-1(a)).[Protocol]

(ii)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. FIG4.a 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 fig4-4(b) are the accumulated PHB in the cells was. 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 (fig4-4(b)), no remarkable fluorescent area was observed.[Protocol]

Perspective

The achievement of our project “Organic synthesis of PHA” is that we registered available PHA synthetic gene in Biobrick parts. 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.

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