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.

A. 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.

B. 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

C. 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

D. 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 show that TB medium is 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 synthesizes the polymer in maximum content rate. However, the growth of E.coli in 37°C is worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C doesn’t make a significant difference. Even if there is no glucose, E.coli synthesizes polymer (condition 9 & 10). We think that TB medium has glycerol and a lot of yeast extra, then E.coli may use them as carbon sources. In addition, the comparison of condition 4 & 5 indicates PA-Ca is not used as carbon sources. LB medium doesn’t contain many carbon sources, so E.coli synthesizes little polymer. In this case, adding PA-Ca doesn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step is 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]

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

[Back to "Construction of phaC1-A-B1 in Biobrick format"]

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]).

[Back to "Construction of phaC1-A-B1 in Biobrick format"]

Protocol

A .P(3HB) production on colonies and preparation before confirmation with Nile red under UV

[Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]

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

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

[Back to "4-1 Confirmation of P(3HB) synthesized on colonies"]

B.P(3HB) production in cells and preparation before the confirmation with Nile blue A

[Back to "4-2 Confirmation of P(3HB) 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

C. Preparation for GC/MS

[Back to "4-3 Confirmation of P(3HB) by GC/MS"]

1. Put 10mg of dried cells in glass tubes.

2. Add 2ml MeOH (containing 15% sulfuric acid) and 2ml chloroform.

3. Incubate tubes at 100℃ for 140min.

4. Add 1ml pure water, stir tubes, and incubate until the solution became clear.

5. Remove the organic layer, filtered.

6. Add internal standard fluid to organic layer.

7. Set in GC/MS.

D. Optimization of the best culture condition to synthesize P(3HB).

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1 Preparing

2 Polymer producing media

3. Culture and collection

3.1 Use LB medium to preculture transformed media 1.5 ml for 15 hrs, 180 rpm/ 37℃.

3.2 Culture 15 μl preculture media into different conditions for 48 hrs, 180 rpm.

3.3 Collect cells and centrifuge for 3 min, 5,000 rpm.

3.4 Remove supernatant and suspend with pure water.

3.5 Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.

3.6 Freeze in -20℃.

3.7 Freeze-dry for 72 hrs.

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