Team:BHSF/Proof Of Concept
Proof of Concept
We first made the solid LB culture medium by mixing NaCl, tryptone, yeast extract, deionized water, agar, and antibiotics
(Amp, Chl, Kan). We then extracted 4 kinds of plasmids that we need.
Then, we prepared the competent cell. We firstly selected BAP1 single colony in 3ml LB medium culture, and added it to 40 ml liquid LB medium culture.
Then, we transferred the bacteria solution to 50 ml centrifuge tubes, and placed them on ice for 10 min. After centrifuging the liquid, we poured out
the culture liquid at clean bench and added the bacteria to 10 ml pre-cooled 0.1 mol/L CaCl2 solution. Bathing them in ice and then centrifuged the solution.
Lastly, adding 2 ml pre-cooled 0.1 mol/L CaCl2 solution to the germ, blowing away the cell. Thus, we successfully expanded the competent cells.
After we constructed the competent cells, transform plasmid BAP1
In order to successfully produce carminic acid, we need the polyketide synthase translated from AntDEFGB, C-glucosyltransferase translated from GtCGT,
cyclase translated from ZhuIJ, and monooxygenase translated from DnrF, to produce carminic acid. We cultured bacteria carrying these kinds of genes.
Then, we expanded the amount of E.coli to make glycerin bacteria and extracting their plasmids. Applying the plasmid to PCR, increasing the number of
antD, antE, antF, antB, antG, and ZhuIJ, purifying them and used gel-electrolysis to justify all the sequences. Using nanodrop to determined the amount.
Besides GtCGT-AmpR and ZhuIJ-KanR, gene antBDEFG, and DnrF is added to the plasmid through same procedure.
Induced Expression
In order to induce the expression of ZhuIJ, DnrF, and GtCGT, we firstly prepared liquid IPTG that was filtered and sterilized.
Expanding the bacteria until the A550 is around 0.6 to 0.8. Adding IPTG until the concentration reaches 0.5mmol/L to the culture medium,
we then induced the bacteria. After induction, we used the E.coli to extract proteins we need.
In order to justify whether our induction is successful or not, 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. After the separation glue is configured, immediately pour it
into the electrophoresis tank between the two glass plates, and then add a thin layer of water and waited for the glue to condense naturally.
After condensation, pour out distilled water to absorb the water. Lastly, equipping with 4% condensation glue.
Then we started to connect the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis and
added the samples into the separation tank. When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis.
Immersing the gel in dyeing solution for half an hour, then decolorize it until the strip is clear.
From the photos above, we know that we successfully expressed antBDEFG and DnrF, However, after 3 times of trying,
we are not able to express protein ZhuIJ, which should be 18.7kD plus 27.3kD.
The principle of our experiment is to use Acetyl-CoA and Malonyl-CoA within E.coli, the polyketide synthase translated from GtCGT,
cyclase translated from ZhuIJ, and monooxygenase, C-glucosyltransferase translated from DnrF, to produce carminic acid. Therefore, if we successfully
expressed those proteins(enzymes), we can surely produce carminic acid through synthetic biology.
Further improvement
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 for induction of protein may inhibit the expression of protein. In the future, we can repeat this procedure in different kinds of E.coli
that can tolerate the toxicity of IPTG, and shorten the time for induction.
Since the usage of carminic acid is mostly abundant, we need to look into how to simplify and increasing the amount of production.
