Revision as of 23:26, 28 October 2013

Constitutive phaCAB

This part has a strong E.coli constitutive promoter in front of the phaCAB PHB biosynthetic operon. Nile Red stain, under UV, shown PHB accumulated inside the cells:

Optimised bioplastic producing operon

In R. eutropha cells, P(3HB) is made through 3 steps. Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB) [2]. We cloned the constitutive promoter J23104+RBS B0034 upstream of the phaCAB operon, replacing its native promoter. Thus this part consists of an anderson constitutive promoter and the phaCAB operon, which function as an optimised bioplastic producing operon.

See our expanding Bioplastic production parts collection: Hybrid phaCAB, Constitutive phaCAB, and Native phaCAB.

Production of P(3HB): Nile Red Staining

O/N cultures of MG1655 transformed with either control (empty vector), native, constitutive or hybrid phaCAB constructs were spread onto LB-agar plates with 3% glucose and Nile red staining.

Production of P(3HB): Microscopy

Streaking the bacteria on Nile Red plates cannot be used to accurately decide the difference between P3HB production due to the effect differing thicknesses of cell layers will have on fluorescence intensity. We imaged the cells using the fluorescent microscope to qualitatively understand how much P3HB is produced per cell. As indicated by other authors, we saw that P3HB is found in localised granules within the bacteria. Empty vector containing cells show little fluorescence. In those that do, the staining is qualitatively different than in the phaCAB containing strains. Nile Red is also used as a lipid stain and this is indicated by its staining of the membranes of EV cells. In contrast, in phaCAB containing cells, staining is restricted to points within the cells.

Each fluorescent image was created by exciting the stain at 530nm for 100ms. Despite this standard treatment of samples the intensity of fluorescence produced by the cells containing the hybrid promoter is greater than from the constitutive ones. This could indicate a difference in the strength of the promoters which lead to phaCAB expression and therefore the amount of P3HB produced. The images also suggest that not every single E.coli is stained. This may be due to the conditions in which the cells were grown or may be dependent upon the stage in the lifecycle of the E.coli.

Fluorescent microscope images of E.coli stained with Nile Red. 1 -Empty vector, 2 - constitutive promoter phaCAB, 3 - hybrid promoter phaCAB. Images labelled a are fluorescent images excited at 530nm ovelaid on the bright field images shown in the b set of images. Images by Imperial College iGEM Team

Comparison of P(3HB) production: constitutive vs. hybrid

The difference in staining between the constitutive and hybrid bacteria is suggestive of increased P(3HB) in the hybrid strain. This is the case. When plastic is extracted from the two strains, more is produced by the hybrid than the constitutive.

P(3HB) at the end of extraction from hybrid and constitutive cells.

Here is a link to the Hybrid phaCAB.

The operon promoted by the hybrid promoter produces more plastic than the construct with the constitutive (anderson J23104) promoter, which in turn produces more than with the native promoter.

• Due to time constraints these bacteria were incubated for a shorter time than those which gave the results on the hybrid biobrick page, therefore these results are not comparable with the ones on that page.

Production of P(3HB): Nile Red Staining

O/N cultures of MG1655 transformed with either control (empty vector), native, constitutive or hybrid phaCAB constructs were spread onto LB-agar plates with 3% glucose and Nile red staining.

phaCAB P(3HB) synthesis constructs transformed into MG1655 Strains were grown on Nile red plates, which stain the PHB strongly and fluoresce in presence of PHB. On the left are MG1655 cells with an empty vector (no fluorescence; no plastic), at the bottom is the native promoter (i.e. low fluorescence, some plastic). At the top and right we have our constitutive and hybrid promoter (respectively), which both show high expression and thus fluoresce very clearly. Imperial iGEM data

Conclusion: The red staining indicates the production of P(3HB). More importantly our new Biobricks hybrid promoter phaCAB BBa_K1149051 and constitutive phaCAB BBa_K1149052 produce more P(3HB) than the native phaCAB operon To find more information about the reasons for improvement, the design and methods of changing the promoter on Imperial iGEM wiki: [http://2013.igem.org/Team:Imperial_College/Waste_Degradation:_SRF Module 1: Waste to bioplastic]

Characterisation

phaCAB biobrick

MG1655 in LB with plasmid EV and the native, constitutive and hybrid promoters for phaCAB. The plasmids all show similar growth curves in LB for all 3 promoters. Indeed this is confirmed by an ANOVA test, which shows no significance in terms of growth difference the constructs (F 3,24 = 0.3573, p < 0.7843). Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Sole Carbon Source

Glucose as a sole carbon source in M9S

phaCAB growth in M9S supplemented with glucose. Constitutive phaCAB grows more at higher glucose concentration of 167 mM while the hybrid grows better at a lower glucose concentration of 22 mM. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Growth on 3HB as sole carbon source

3HB sole carbon source growth in M9S with P3HB synthesis pathway genes. The 3 phaCAB promoter constructs are represented with native phaCAB, constitutive phaCAB and hybrid phaCAB. In addition bdh2 was tested to see whether its presence increased growth for the cells. This was completed with 3 3HB concentrations - 100 μM, 10 mM and 100 mM. ANOVA analysis shows that there is no significant difference between any of the constructs (F 4,10 = 0.1875, p < 0.9396). Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Growth on acetoacetate as sole carbon source

phaCAB promoter constructs in M9S with acetoacetate as sole carbon source. Empty vector shows the least growth of all the constructs of MG1655. The best performing construct is hybrid phaCAB. According to ANOVA analysis, there is no statistical significance between the different phaCAB promoter constructs or EV (F 3,12 = 3.086, p < 0.068) with regard to growth on acetoacetate as a sole carbon source. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Growth on sole carbon sources

Constitutive promoter in phaCAB growth in sole carbon sources under M9S. MG1655 E. coli transformed with native phaCAB compared to empty vector for sole carbon sources. The trends observed show that there is not much difference between EV and phaCAB for glucose (p = 0.6511), acetoacetate (p = 0.4633) and 3HB (p = 0.7266). ANOVA analysis concluded that null hypothesis, "The sole carbon sources tested have no effect on growth" must be rejected (F 5,12 = 15.94, p < 0.0001). This shows that there is a difference between individual carbon sources. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Partial sequence of the constitutive phaCAB construct (BBa_K1149052):

References

References 1. H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010) 2. Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).

Sequence and Features