Difference between revisions of "Part:BBa K3192031"

Latest revision as of 00:54, 22 October 2019

pha plasmid with neokanamycin resistance

Production of P(3HB)

The 2019 Virginia iGEM team combined the genes phaA, phaB, and phaC, endogenous to Cupriavidus necator and codon optimized them for E. coli K12 strains. Before each gene a custom synthetic ribosomal binding site was inserted to optimize translation initiation rates of each gene. The 2019 Virginia iGEM’s addition to the T7 registry (BBa_K3192012) was used as the inducible promoter that was regulated by the presence of IPTG. IPTG added to the growth media induced high levels of transcription and translation to express the coding sequence of phaABC (For more information about the promoter see its part page).

The genes phaA, phaB, and phaC encode for enzymes essential for the conversion of acetyl coa into polyhydroxybutyrate (PHB). phaA (BBa_K3192015) encodes for b-Ketoacyl-CoA thiolase. phaB (BBa_K3192016) encodes for b-Ketoacyl-CoA reductase. phaC (BBa_K3192017) encodes for poly(3-hydroxybutyrate) polymerase. Below the biochemical pathway associated with these genes is displayed below.

This figure demonstrates the pathway for the production of PHBs from acetyl-CoA using the enzymes encoded by the gene cluster phaACB.

This plasmid was expressed in E. coli TG1 cells, and grown in M9 minimal media. M9 is an idea culture media for PHB producing bacteria because it is nitrogen limiting. The absence of nitrogen in media drives the production of PHBs within cells. Cultures were grown for 72 hours at 30℃, as it was indicated that 30℃ was more optimal for PHB production in experiments performed in 2018 by Johnston et al found produce greater yield when cultured under these conditions.

Use of Beta Neo-kanamycin to dual plasmid system

Keeping two plasmids in a culture that is continually growing can sometimes be difficult. Ensuring that both plasmids are doubled and split equally on fission is important. To do this, the 2019 Virginia team used a split antibiotic system developed by iGEM 2017 Vilnius’s plasmid control design. Having both plasmids contain a gene to produce a partial protein for kanamycin resistance, and combining together to form a full resistance complex was the goal of the plasmid design. This would regulate resistance towards the bacteria that weren’t capable of producing both plasmids, and ensured that the resistance would continually select for those that maintained both plasmids during fission. The kanamycin construct would hypothetically be more efficient for long-term culturing of bacteria, in terms of stifling the effects of evolution for a longer time than would a dual resistance system.

The BioBrick BBa_K2259019 developed by Vilnius in 2017 contained resistance for half of the kanamycin antibiotic. Virginia iGEM used this BioBrick to maintain this part in the same chassis as BBa_K3192029, which possessed the BioBrick BBa_K2259018, conferring the second half of the kanamycin resistance.

Red Nile Staining

Our team successfully constructed a plasmid that is capable of producing PHBs given simple carbon sources, such as glucose, which we used for preliminary functional testing of our BioBrick’s (BBa_K3192031) function. To determine qualitatively if BBa_K3192031 worked, we performed Red Nile staining to test for PHA presence.

Red Nile staining of E. coli K12 expressing BBa_K3192031 which produces PHBs.

E. coli K12 cells expressing no genes for PHB production (negative control).

PHB Extraction Method

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 cells expressing BBa_K3192031 to quantify yield. Through sonication and separation by density differentials our team extracted 0.0427 g of PHB from a 200mL culture growth for 72 hours at 30℃. This protocol is still being further developed to increase yield and purity of PHB.

Extraction of PHBs using density differentials and sonification

1) Transfer cell culture into a 500 mL centrifuge bottle.
2) Centrifuge at 5000 for 20 min.
3) Remove supernatant (pour off if pellet is condensed enough) and resuspend in ~15mL of water
4) Decant the supernatant and resuspend the cell pellet in ~20mL of Milli-Q water. Pipette the resuspended cells into a sonication tube.
5) 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)
          5-7x sonication for 30 with 15 sec in between (rest prevents over heating)
          0-80% 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

Sequence and Features