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<h2>Notebook</h2> | <h2>Notebook</h2> | ||
Revision as of 04:06, 19 December 2020
Categories
Result
1. Optimize and synthesis the sequence of four fusion proteins (M7824-CILY, M7824-SILY, M7824-Type4, M7824-Type4(reversed).
2. Realize the microcellular expression of four fusion proteins(M7824-CILY, M7824-SILY, M7824-Type4, M7824-Type4(reversed).and PDGFβR-TRAIL.
3. Realize the prokaryotic expression of four fusion proteins.
4. Assess cell model successfully with PDGFβR-TRAIL.
5. Prove the genes (collagen I, α-SMA etc.) expression difference with RT-qPCR.
6. Prove the collagen I deposition difference with Immunocytochemistry (ICC)
Sequence Synthesis and validation
As we extracted the domains from human genome, it’s not that convenient for cell-free expression or prokaryotic expression. With the technical support from Sangon, we successfully optimize the condons of 4 recombinant proteins.
As we need to verify our composite sequences ,we designed specific primers then verified it by PCR and double enzyme digestion. And thanksfully, we prove that our sequences were perfectly synthesized.
Fig1. PCR results after double-enzyme digestion
Cell-free Expression of selected peptides
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability.[1] Thus, CFPS enables direct access and control of the translation environment which is advantageous for a number of applications including co-translational solubilisation of membrane proteins, optimisation of protein production.
Since we wanted to choose a quick and easy way to rapidly produce a certain amount of proteins for preliminary molecular verification tests and cell verification tests, CFP became our best choice, and we are very proud to be the first team to use this technology in the iGEM competition.
Expression template construction
Fig2. The schematic diagram of our sequence for cell-free expression
To achieve it ,we designed special primers for template reconstruction,
Table1 Primers for adaptor insertion
Then we mixed and spliced fragments 1, 2 and 3 according to 1:1:1. Lane 1 is the result of SILY stitching, lane 3 is the result of CILY stitching, lane 4 is the result of Type4R stitching, lane 6 is the result of Type4 stitching, and lane 8 is Marker. The glue was recovered with the correct size of the strip, and the concentration was determined for cell-free expression.
Fig3. Stiching result
Then, we purified fragments and added them into a 5mL cell-free expression system at a final concentration of 5ng/uL, and expressed at 26℃ and 150rpm overnight.
After purification and concentration, we did SDS-PAGE to verify their successful expression. As we are not satisfied with its yield, we are consisting on testing different expression conditions to get better output.
Fig4. Cell-free expression result
Prokaryotic expression of selected peptides
(1)Expression of proteins we designed
Firstly, we used E.Coli (BL21) with DH5-a to amplificate the plasmids inserted with our sequence.Then, we used E.Coli (BL21) with pET28a then expanded its culture to express adequate proteins. We take two steps to verify their expression.
Fig5.Direct SDS-PAGE
Though we have seen the expression of our proteins in SDS-PAGE, we need to expand our output so that we expanded its culture to 1.5L~2L, and extracted proteins then purified them.
Fig6. Purification and extraction process
Fig7. SDS-PAGE after expanding cultivation
As our experiment time is limited, we decide to assess our cell model with PDGFβR-TRAIL ( a functional protein which can block PDGF relating pathway, because of which Prof. Yang Hao generously wanted us to assess its function of resisting pulmanory fibrosis)
Fig8.SDS-PAGE result of PDGFβR-TRAIL expression
Function Verification in HFL-1 Pulmonary Fibrosis Model
We successfully establish a pulmonary fibrosis model with HFL-1 (treated by TGF-β1) then set nine groups
and apply RT-q PCR to determine whether their epithelial-mesenchymal transition (EMT) process was blocked with the level of collagen I.
Fig9. DMEM+10%FBS,untreated,48h(20x)
It can be obviously refered from the cell morphology that, the HEL-1 cells differentiated into myofibroblasts in the TGE-beta-treated group while this process was depressed in the drug-treated group.
To get more evidence to prove our cell model and drug treatment success, we used RT-qPCR to get more evidence in gene expression degree.
We can see that it occurs expression degree of collagen I differences between three groups though the difference is not that ovious. But we are making more drug concentration comparisons to detect the differences more obviously, and we believe that these experiments will provide guidance for later SPR experiments and animal experiments in the future.
Notebook
https://2020.igem.org/File:T--SCU-WestChina--Notebook.pdf
