Team:Scut-Champion-Park/Design
In order to solve the problem of food safety caused by the abuse of antibiotics, our group constructed the tetracycline inducible expression system. And through plasmid mediated, the system was transfected into Escherichia coli TOP10 and GS115 Pichia pastoris. It can generate a rtTA transcription activation factor in cells. RtTA combining with tetracycline antibiotics can activate the fluorescent protein expression system, which can instructs the tetracycline class of antibiotics by fluorescence detection.
rtTA: combined with rTetR (reverse TetR) and VP16 transcriptional activation of regional. RTetR derived from the mutation of the 4 amino acids in TetR.
The fluorescent protein expression system: TRE (Tet-responsive element), PminCMV (minimal CMV minimal promoter) and fluorescent protein gene composed of it.
Tet-on system is widely used in the study of gene function, protein function and mRNA function in vivo and in vitro of animal. It also used in gene therapy. Its characteristic is low background and high induction ratio. Its induction ratio can reach 10000 times.
The Tet-on system has two parts, they are regulation plasmid and expression plasmid. They are all constructed by us. The rtTA gene can express rtTA protein. When there is tetracycline, it will change its configuration and can combine with TRE which is the regulation sequence of the promoter. PminCMV is the recognition sequence of the promoter. Then the expression plasmid produce fluorescent protein and we can use it to detect the tetracycline class of antibiotics.
Pichia is easy to cultivate. It propagated rapidly and can process, fold and modify the exogenous protein after translation Closer to higher organisms. It will help to genetic engineering operation and high density fermentation.
Combining the advantages of them two, we construct the recombinant Pichia with tet-on system. Use it as a biological detector of tetracycline. It can detect the tetracycline in the environment easily. That will make it easy to detect by customer ourselves and help to build consumers' confidence.
(1) We construct a simple biological detector using microorganism by the method of synthetic biology and can detect the tetracycline.
(2) The detector can tell us if there is tetracycline by obvious signal, playing the role of biological test paper.
(3) It is easy to operate and has important significance for food safety.
(4) Engineering bacterium can be fermentable on large scale. This will make it cheaper than other methods.
The design and construction of Tet-on regulation plasmid
The design and construction of Tet-on expression plasmid
Preparation of recombinant yeast and the Transformation and screening of recombinant yeast
We design the primer by software and get the rtTA gene from Tet-on plasmid. Add EcoR1 and Not1 on the ends of it. We reconstruct the system because the original system is not apply to yeast.
To ensure the success of the experiment, we remoded the Plasmid. We link the rtTA or rtTA-flag with Restriction Enzyme cutting site to the Plasmid pGAPZB. Construct the Plasmids pGAPZB-rtTA and pGAPZB-rtTA-flag.
The Plasmids are led to the GS115 bacterial strain which are HIS auxotroph. Under The effect of GAP promoter, The bacterial can produce rtTA protein. In other word, we construct the regulation system in yeast and screen by resistant plate.
On the base of pTRE2, we determine the modification conditions of Tet-responsive promoter by correlated references. Use the CYC1TATA frame structure in yeast and construct the TRE-CYC1TATA promoter by fusion PCR. The promoter can sensitively regulate the Tet-On system in yeast. Link the Resistance selection markers such as HIS4 and Kanamycin by clone, we construct the pPICTC-EGFP Plasmid.
Lead the pPICTC-EGFP Plasmid into the competent Yeast with pGAPZB-rtTA or pGAPZB-rtTA-flag to complete the construction of recombinant yeast.
Prepare recombinant yeast by chemical approach. Transform the yeast by electroporation transfection.
Transformation steps:
1) Prepare recombinant yeast by chemical approach, 80μL/tube, use it immediately or conserve it at -80℃.
2) Add 0.1~0.2mg linearization recombinant plasmid into the recombinant yeast cells, then take them in the 0.2cm electric shock cup in the ice-bath for 5min.
3) Electric shock it by pulse cell transfection system at 1.5Kv, 200Ω, 25mF, 5ms.
4) Add 1ml 1mol/L sorbitol taken from ice-bath into the mixed liquor after electric shock. Take it to 1.5ml centrifuge tube and wait for 1.5h at 30℃.
5) Take 200μl bacterium solution, smear it on the Resistance or auxotroph plate to screen.
Principle of electroporation transfection:
We lead the linearization plasmid into the inner of yeast cell. The linearization plasmid will occur homologous recombination with the genome of yeast to lead our target gene into the genome of yeast. We can screen the transformant by cultivate the cell on resistance or auxotroph plate.
Verification of detecting the tetracycline in food and measure the detection limit, comparing with existing methods.
At last, we compared our method with traditional methods such as HPLC and ELISA to identify that our method is feasible.