Fig2-2-1-1, Rose silhouette on the LB agar plate containing Nile red.

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

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] )

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]

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 product as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry. To confirm 3HB by GC/ MS, 3HB is methylated because 3HB is difficult to measure. Following Fig2-2-4-3-1, the peaks of sample are same to those of standard control of methylated 3HB. This shows E.coli synthesized P(3HB) correctly. [Protocol]

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

4-4 Make purified P(3HB) sheets

We made P(3HB) sheets. To make the sheets, we cultured E.coli JM109 in erlenmeyer flasks at 37°C for 72h. Contact angle is an indicator to represent the strength of the water-repellent. The angle shows the physical properties, especially surface tension. When contact angle of sheets is larger than 90°, from Young equation, the sheets would have more strong water-repellent by increasing real surface area. Contact angle of P(3HB) sheets is about 100° from literature data. [Protocol]

Fig2-2-4-4-1, P(3HB) sheet

4-5 Examine best culture condition to synthesize P(3HB)

To examine best culture condition, we tried culturing E.coli JM109 in 10 conditions for 48h. This culture is done in test tubes. Composition of LB and TB medium is shown in Fig2-2-4-5-1.

Fig2-2-4-5-1, Composition of LB & TB

Fig2-2-4-5-2, Structure of Pantothenic acid

Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA. The culture results are shown in Fig2-2-4-5-3.

Fig2-2-4-5-3, Culture results of ten conditions

In Both LB and TB, when we added glucose and PA-Ca, culturing at 37°C, we got maximum polymer content rate. However, the growth of E.coli in 37°C is worse than that of in 30°C, so final polymer concentration in 37°C and 30°C doesn’t make significantly difference. Even if there is no glucose, E.coli synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so E.coli may use them as a carbon source.

In addition, according to condition 4 & 5, PA-Ca is not used as a carbon source. Since E.coli has rich carbon source in TB medium, according to condition 7 & 8 and 9 & 10, the glycolytic pathway may become a rate-limiting step, so polymer production would be increased by adding PA-Ca.[Protocol]

Application

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

The achievement of our project “P(3HB) Production” 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

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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.

[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