Latest revision as of 01:22, 22 October 2019

phaC1-A-B1 [P(3HB) synthesis]

Imperial College London 2013 Experience

Increased production of P(3HB)

The Imperial College iGEM team have successfully purified P(3HB) from E. coli. (MG1655) transformed with either native phaCAB (BBa_K934001) or hybrid promoter phaCAB (BBa_K1149051). Our novel Biobrick hybrid promoter phaCAB (BBa_K1149051) produces significantly 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/BioPlastic_Recycling:_PHB PHB recycling.]

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

A summary of the improved production of P3HB by our hybrid promoter-phaCAB construct(BBa_K1149051) over the native promoter-phaCAB. Imperial iGEM data

Comparison of P3HB production P3HB extracted from E.coli MG1655 transformed with left to right, control (empty vector), native phaCAB (BBa_K934001) and hybrid promoter phaCAB (BBa_K1149051). Each produced in 300ml cultures of LB with 3% glucose after one night growing at 37 degrees celsius. Imperial iGEM data

Comparison of P3HB production <b>(left) 1.5ml tube, natural phaCAB (BBa_K934001) (right) 5ml tube, phaCAB expressed from the hybrid promoter, (BBa_K1149051). Imperial iGEM data

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

Sole Carbon Source

Use of glucose as a sole carbon source in minimal media:

phaCAB growth in M9 minimal with 3% glucose sole carbon source. LB grown MG1655 phaCAB grow more rapidly initially then M9M with 3% glucose sole carbon source but reach the same OD after 6h while EV shows a different trend. EV in M9M levels off at a much lower OD at 4h, as seen with EV grown in LB. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

phaCAB growth in M9S supplemented with 3% glucose. LB grown MG1655 phaCAB grow more rapidly initially then M9S with 3% glucose sole carbon source, but after 5h, phaCAB in M9S continue to grow to a higher OD. EV shows a different trend, in M9S it levels off at a similar level to LB. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Use of 3HB as a sole carbon source in minimal media:

3HB sole carbon source growth in minimal media with phaCAB and EV. MG1655 phaCAB were grown in supplemented and minimal M9 media. Relative to the empty vector control, there was no significant difference in growth as t-test gave p = 0.8072 > 0.05. However, an ANOVA of the data gave (F 3,8 = 6.589, p < 0.0149), thus the null hypothesis, there is no difference between M9M and M9S must be rejected. Indeed if we look closer, we see that both M9M EV and M9S EV are significantly different from another (p = 0.045), as are M9M phaCAB and M9S phaCAB (p = 0.0284). 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.

Use of acetoacetate as a sole carbon source in minimal media:

phaCAB in M9 minimal and M9 supplemented media with acetoacetate as sole carbon source. Both media have a similar trend with increased acetoacetate concentration. Of interest is phaCAB in M9S at 10 mM, as this appears to be a local optimum, indeed this is seen in M9M grown phaCAB, while empty vector at 10 mM grows much less in comparison. An ANOVA for this data gives (F 3,12 = 1.669, p < 0.2262), which means that there is no significant difference between the data. 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.

Use of acetoacetate, glucose and 3HB as a sole carbon source in M9 minimal media:

phaCAB growth in sole carbon sources under M9M. M9 minimal grown with phaCAB shows that 3HB is a poor carbon source at all concentrations tested. In addition, acetoacetate is poor as well at low concentrations, however at 10 mM acetoacetate, there is a spike in growth whereby it outperforms glucose. Glucose itself permits strong growth, especially as the concentration is increased from 22 mM to 167 mM. This said, ANOVA analysis showed that there was no significant difference between these values (F 2,6 = 2.745, p < 0.1424). 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.

Use of acetoacetate, glucose and 3HB as a sole carbon source in M9 supplemented minimal media:

phaCAB growth in sole carbon sources under M9S. M9 supplemented media grown with phaCAB displays similar results to those seen in M9M media. 3HB is once again the poorest carbon source, while acetoacetate has an optimum at 10 mM. Glucose performs very well in this situation as a sole carbon source. When submitted to ANOVA analysis, the sole carbon sources showed significant difference from each other (F 2,6 = 8.622, p < 0.0172). Testing the null hypothesis that there is no difference between sole carbon sources reveals the following. Applying a t-test shows that while 3HB and acetoacetate are not significantly different as carbon sources (p = 0.2341), both 3HB and glucose (p = 0.0256) and acetoacetate and glucose (p = 0.0488) act differently as sole 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.

Use of acetoacetate, glucose and 3HB as a sole carbon source in M9 supplemented minimal media compared to empty vector:

Native 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 = 0.6237), acetoacetate (p = 0.8781) and 3HB (p = 0.9901). ANOVA analysis concluded that null hypothesis, "The sole carbon sources tested have no effect on growth" must be rejected (F 5,12 = 6.938, p < 0.0029). 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.

Toxicity Assays

3HB toxicity:

Toxicity of 3-hydroxybutyrate in LB with phaCAB and EV expression. 3HB was tested at 5 concentrations for toxicity - 1 μM, 100 μM, 1 mM, 10 mM and 237 mM. Between 10 mM and 237 mM, there is a clear toxicity effect observed. 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.

Acetoacetate toxicity:

Toxicity of acetoacetate in LB with phaCAB and EV expression. Acetoacetate was also tested for toxicity at 4 concentrations - 1 μM, 100 μM, 1mM and 287 mM. In phaCAB and EV this manifested itself between 1 mM and 287 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.

P(3HB) toxicity:

Toxicity of Poly(3-hydroxybutyrate) in LB with phaCAB and EV expression. P3HB toxicity was only tested at one concentration in LB - 31 μg/L, it was also tested at 31 mg/L however the resulting OD was over 1, so this data was not considered accurate. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

PLA toxicity:

Toxicity of Poly(lactic acid) in LB with phaCAB and EV expression. Polylactic acid in the form of ground up plastic did not show any toxicity effect at 31 mg/L in LB. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

BNU-China 2016 Experience

In order to make the P(3HB) production process more controllable, we added heat-sensitive promoter(BBa_K873002) and arabinose inducible promoter(BBa_I0500)to phaC1-A-B1 gene sequence (BBa_K934001), constructing the heat and arabinose inducible parts (BBa_K1891014) and (BBa_K1891015). Finally we linked the newly synthesized parts to the standard vector pSB1C3. The new plasmids can be used directly for heat shock and arabinose induced expression in E.coli.

We transformed the constructed vectors to TransB(DE3) for expression, and set different conditions for inducement.

The results show as follow,

A,B left to right: pSB1C3 empty(37℃)，pSB1C3 empty(42℃ induced)，P(3HB) with natural promotor，P(3HB) with HSP 1,2(37℃)，P(3HB) with HSP 1,2(42℃).
C,D left to right: pSB1C3 empty(37℃)，pSB1C3 empty(Arabinose induced)，P(3HB) with natural promotor，P(3HB) with pBAD 1,2(37℃)，P(3HB) with pBAD 1,2(Arabinose induced)

iGEM12_Tokyo_Tech

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. As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.

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 poly-3-hydroxybutyrate, 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.

We cultured the transformant on LB agar medium plates with 0.5μg/ml Nile red and 2% glucose at 37℃ for 30 hours, then we transferred the plates to 4℃ room. After 115 hours, colonies with PHB would be stained red by Nile red when observed under UV.

FIG1.A: E.coli JM109 colonies with BBa_K934001 gene, PHB accumulation. FIG1.B: E.coli JM109 colonies with PlasI-gfp gene, no PHB accumulation.

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.

We cultured the colony in LB solution for 16hrs at 37℃, then we concentrated the solution and painted the letter by the solution on LB agar medium including 0.5μg/ml Nile red and 2% glucose at 37℃ for 36 hours.

FIG2 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.

Using the LB solution, we painted a rose silhouette on the LB agar plate containing Nile red. (FIG3).

FIG.3 Rose silhouette on the LB agar plate containing Nile red.

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.a are the accumulated PHB in the cells was. This result also indicates that part BBa_K934001 synthesized PHB. In the photograph of negative control (FIG4.B), no remarkable fluorescent area was observed.

To take this photo we did shaking culture at 37 ° C for 96 hours. Then, we froze dry the cells and stained them by Nile blue A.

FIG4.A E.coli JM109 cells with PHB accumulation).FIG4.B E.coli JM109 cells without PHB accumulation. Optical magnification X100).

For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#3. our work in Tokyo_Tech 2012 wiki].

Virginia 2019 experience

The 2019 Virginia iGEM team was focused on PHB production and decided to explore a few quantitative and qualitative aspects of this plasmid.

Our team performed a preliminary Red Nile staining of the cultures after 72 hour culture of K12 DH5 E. coli at 37°C in M9 minimal media. The choice of M9 as opposed to LB used by Tokyo Tech was due to the ideal nitrogen limiting conditions of the media, which is a proven factor to influence PHB production.¹ 72 hours was also cited as the culture time for maximum PHB/cell yield.

Our team also wanted visual insight of PHB production in a cell on the cellular level. With the help of the University of Virginia Medical School Microscopy Center our team gather Electron Microscopy images of the production of PHB by cells expressing BBa_K934001. The EM pictures were not obtained in time for the parts registry freeze, however our team plans to display the images at the Jamboree.

To characterize and quantify the production of PHBs from part BBa_K934001 our team performed Red Nile staining to determine if PHBs were produced under these conditions. As can be seen by Red Nile results from a non PHB producing control and the BBa_K934001 expressing cells, PHBs were indeed produced through 72 hour cultures in M9 minimal media.

Red-nile staining of cells without any PHA-producing plasmid (A), and cells that contain the iGEM part BBa_K94001 (B).

The 2019 Virginia iGEM team also developed a method of PHB extraction with lower environmental impacts detailed below. Our method involves sonication of cells to lyse, and sucrose solution to separate PHB from cell pellet using density differential. This eliminates the use of chemicals that are harmful to human health and the environment. Normal PHB extraction involves potent chemicals such as chloroform, methanol, and sodium dioctyl sulfate are not easily disposed of in lab settings, and therefore are more environmentally costly to do so. We performed this extraction method on our BBa_K934001 cells to quantify yield. Through sonication and separation by density differentials our team extracted 0.0427 g of PHB from a 200 mL culture grown for 72 hours. This protocol is still being further developed to increase yield and purity of PHB.

PHB pellet (bottom pellet) and cell pellet (top pellet) separated by a sucrose cushion of 1.23 g/mL. Separation performed by differential centrifugation.

Additionally, the 2019 Virginia iGEM team explored the use of custom Synthetic Ribosomal Binding Sites to increase translation initial rate of our devices. We analyzed the translation initiation rates of the three RBS sites of BBa_K934001 using DeNovo software. The overall translation initiation rate of this device was run through thousands of iterations and found to be 429. This is an arbitrary unit but initiation rates are desired to be in the range of 50,000, indicating that the use of synthetic ribosomal binding sites could increase the efficiency of expression for BBa_K934001.

Extraction of PHBs using differential centrifugation and sonication

Transfer cell culture into a 500 mL centrifuge bottle.
1) Centrifuge at 5000 for 20 min.
2) Remove supernatant (pour off if pellet is condensed enough) and resuspend in ~15mL of water
3) Decant the supernatant and resuspend the cell pellet in ~20mL of Milli-Q water. Pipette the resuspended cells into a sonication tube.
4) Sonicate cells 5-7 times, or until about 80% of the cells are lysed (observe cells under microscope). Note: This part is empirical and can vary greatly depending on cells Use flat tip (larger tip)
5) 5-7x sonication for 30 with 15 sec in between (rest prevents over heating) 70-80% amplitude
6) Prepare sucrose solution with a density of 1.2g/mL
7) Add sucrose solution to cells and PHB pellet
8) Centrifuge at 6,500 rpm for 15minutes at 6C in SS34 rotary
9) Remove supernatant, leaving only PHB below sucrose solution
10) Resuspend PHB pellet in water
11) Let dry overnight until all water has evaporated

References:

1) Johnston, Brian, Radecka, Iza, Hill, David, … Marek. (2018, August 29). The Microbial Production of Polyhydroxyalkanoates from Waste Polystyrene Fragments Attained Using Oxidative Degradation. Retrieved from https://www.mdpi.com/2073-4360/10/9/957.

Sequence and Features