Team:BHSF/Engineering
DESIGN
The design of our experiment is to use Acetyl-CoA and Malonyl-CoA within E.coli, the polyketide synthase translated from AntDEFGB, C-glucosyltransferase translated from GtCGT, cyclase translated from ZhuIJ, and monooxygenase translated from DnrF, to produce carminic acid. Therefore, if we successfully expressed those proteins(enzymes), we can surely produce carminic acid through synthetic biology.
Plasmid Construction and Test
BUILD
LB culture medium
After mixing all the substances, the mixture is placed into the autoclave to be sterilized. After cooling it down, we added antibiotics( Amp, Chl, Kan) to the mixture, since they cannot tolerate high temperature. Pouring the liquid mixture to culture dishes, and waiting until they become solid.
Plasmid extraction
The kits provide all we need to extract these 4 kinds of plasmid: pET28a, pACYC, pYES2, and pCDF. After adding solution of bacteria E. to centrifuge tubes, they are centrifuged with 12,000 rpm/min for approximately 2 min, in order to remove the supernatant liquid. Following the instruction of the kit, our members successfully extracted 4 kinds of plasmid from E.coli.
Transformation of plasmid
Firstly, BAP1 single colony was selected in 3ml LB medium culture, and shook over night at 190rpm at 37 ℃. 0.4 ml pre-cultured bacteria solution was transferred to 40ml liquid LB medium culture. They are shook for 2 to 3 hours at 250 rpm at 37 ℃, until OD600 = 0.3 to 0.4. Transferring the bacteria solution to 50ml centrifuge tubes, and placed them on ice for 10 min. The tubes are centrifuged for 10 min at 4,000rpm at 4 ℃. Pouring out culture liquid completely at clean bench, and remember to be gentle and careful. Adding the bacteria to 10 ml pre-cooled 0.1 mol/L CaCl2, gently blow the germ with 5ml pipette. Bathing them in the ice for 30min at 4,000rpm at 4 ℃. Then centrifuged them for 5 min to completely remove their supernatant. Lastly, adding 2 ml pre-cooled 0.1 mol/L CaCl2 solution to the germ, blowing away the cells, and here we got the competent cells to use immediately or divide into parts of 200ul, and freeze them at -80℃ for later use.
In order to successfully produce carminic acid, we need enzymes that are translated by genes called Ant BDEFG, ZhuIJ, DnrF, and GtCGT. After receiving the puncture bacteria carrying GtCGT and ZhuIJ genes, they were cultured on LB plates containing corresponding antibiotics (GtCGT-AmpR and ZhuIJ-KanR)overnight at 37℃. On the next day, selecting single colonies bacteria add to 10ml liquid LB medium culture to expand them at 220rpm at 37 ℃. After expanded culture, single colonies are selected to make glycerin bacteria, extracting their plasmids.
TEST
Applying the plasmid to PCR, increasing the number of antBDEFG, and ZhuIJ, purifying them and used gel glycolysis to justify all the sequences. Using nanodrop to determined the amount. Besides GtCGT-AmpR and ZhuIJ-KanR, gene DnrF and GtCGT is added to the plasmid through same procedure.
Protein Construction and Test
BUILD
Induced expression
First prepare 5ml 1mol/L of liquid of IPTG. After filtration and sterilization, stored at -20℃. Draining 500ul bacteria liquid to 50ml liquid LB culture medium that has corresponding antibodies, 220rpm shaking the culture medium at 37℃ for 2.5 hour, until the A550 is around 0.6 to 0.8. Adding IPTG until the concentration reaches to 0.5mmol/L to the culture medium, inducing them at 37℃ for 4 hours. After induction, we used the E.coli to extract proteins we need. Centrifuging the bacteria solution at 6,000rpm for 10 minutes at 4℃, abandon the supernatant. Adding PBS buffer at least 3 times volume of the bacteria. Using high temperature to break them in ice-bathing condition, until the heavy suspension becomes no viscous liquid. After the centrifugation with 12,000rpm for 20 minutes at 4℃, collecting the supernatant, and preserving them at -80℃.
TEST
SDS-PAGE
Applying the plasmid to PCR, increasing the number of antBDEFG, and ZhuIJ, purifying them and used gel glycolysis to justify all the sequences. Using nanodrop to determined the amount. Besides GtCGT-AmpR and ZhuIJ-KanR, gene DnrF and GtCGT is added to the plasmid through same procedure.
We put the protein extracted from E.coli to SDS-PAGE. Our team instructor helped us prepare the separation gel, protein sample treatment solution, and SDS electrolysis buffer. In gel preparation, we first put the dried glass plate on the support of electrophoresis tank and fix the glass plate, then equipping them with 12% separating glue. After the separation glue is configured, immediately pour it into the electrophoresis tank between the two glass plates, stop about 3cm away from the upper mouth, and then add a thin layer of water for water sealing for about 40 minutes, waiting for the glue to condense naturally. After condensation, pour out distilled water to absorb the water. Lastly, equipping with 4% condensation glue.
Connecting the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis. First, the constant voltage is 80V. After the sample enters the separation gel, the constant voltage is 120V. When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis.
Finally we need to prepare the dyeing and decolorizing solution according to the following diagram.
Dyeing: remove the gel from the electrophoresis tank, carefully remove the gel, immerse the gel in dyeing solution for half an hour.
Decolorization: decolorize until the strip is clear.
From the photos above, we know that we successfully expressed antBDEFG, GtCGTand DnrF, However, after 3 times of trying, we are not able to express protein ZhuIJ, which should be 18.7kD plus 27.3kD.
LEARN
In our experiment, ZhuIJ was not expressed after tried for 3 times. This may because of the toxicity of IPTG for E.coli, also the long time or high temperature for induction of protein may inhibit the expression of protein. In the future, we can repeat this procedure in shorter time and lower temperature.
Since the usage of carminic acid is mostly abundant, we need to look into how to simplify and increasing the amount of production.
