Difference between revisions of "Team:NAU-CHINA/testh"

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Fig4. Function verification of TEV elimination of modified tetR inhibition with eukaryotic cells
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Figure5. Function verification of recombinases in HEK 293T cell
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Stably transfer Jurkat T cells with the modified TetR gene to construct a stably transferred cell line. Then transfer plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes into the aforementioned stably transferred cell. Co-culture the 293T cells expressing GFP on the cell surface with these Jurkat T cells for 4 h when 293T cells were deposited at the bottom of the culture medium and separated from suspended Jurkat T cells.
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Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase recognition sites and corresponding recombinase gene.
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(A) Fluorescence microscope observation of the stably transfferred cell line.
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(B) Fluorescence microscope observation of the stably transfferred cell line transferred plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes.
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The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (upper panel) or transfected with plasmids containing corresponding recombinase gene together (bottom panel), are shown.
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(C) Fluorescence microscope observation of the aforementioned Jurkat T cells co-cultured with 293T cells expressing cell surface-expressed GFP for 4 h.
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Through fluorescence microscopy, we could observe that the suspended T cells emit green fluorescence, which is clearly distinguished from the weaker green fluorescence of 293T cells expressing surface-expressed GFP  deposited at the bottom of the culture medium. The results show that TEV can relieve the inhibition of tetR on the promoter in 293T cells. It means that we have successfully verified the function of TEV - activated transcription system based on the modified tetR in eukaryotic cells and the results also confirm preliminarily that our upstream circuit can work normally. However, we have to admit that due to we chosed GFP as our reporter gene, it is difficult to distinguish it from cell surface-expressed GFP. Our verification experiment is not intuitive. If we need to prove the function of TEV suppressing the inhibition of tetR strongly, further  optimized experiments are still needed.
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The results show that the recombinases can recognise the sites and reverse the sequence between sites in HEK 293T.
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Downstream circuit
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The downstream pathway is the core circuit for us to realize the threshold function. According to literature  [3], they have verified the inversion function of the three recombinases in prokaryotic cells and proved the threshold function of the recombinases, i.e. the recombinases do not have the inversion function at low concentration. Only when the concentration of recombinase reaches a certain threshold, can the recombinases work normally.
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Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells
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According to the same document, we designed our pathway in eukaryotic cells, expecting to realize threshold switching in eukaryotic cells. For this reason, we try to test the inversion function of recombinases and the threshold characteristics of the combination of three different recombinases ( Bxb1, TP901, φ C31 ) and three promoters with different intensities ( miniCMV, EF1 - α, Ubc ) in eukaryotic cells.
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We also verify the function of RDF [4] to demonstrate our 0/1 switch resettable in HEK 293T cells.
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Fig.6. Function verification of recombinases in HEK 293T cell
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HEK 293T cells were co-transfected with six different amounts of plasmids containing recombinase genes (miniCMV-Bxb1 and miniCMV-TP901) , and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co-transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. Transfection of different amounts of plasmids containing recombinase genes into cells indicates that cells can produce recombinase at different concentrations. The experiment was repeated three times.
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(A) Fluorescence microscope observation of HEK 293T undergone different experimental treatments
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(B) The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.
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(C) The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.
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The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.
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Function verification of RDF in HEK 293T cell

Revision as of 08:33, 17 October 2018

Figure5. Function verification of recombinases in HEK 293T cell Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase recognition sites and corresponding recombinase gene.

The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (upper panel) or transfected with plasmids containing corresponding recombinase gene together (bottom panel), are shown.

The results show that the recombinases can recognise the sites and reverse the sequence between sites in HEK 293T.

Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells

Fig.6. Function verification of recombinases in HEK 293T cell HEK 293T cells were co-transfected with six different amounts of plasmids containing recombinase genes (miniCMV-Bxb1 and miniCMV-TP901) , and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co-transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. Transfection of different amounts of plasmids containing recombinase genes into cells indicates that cells can produce recombinase at different concentrations. The experiment was repeated three times. (A) Fluorescence microscope observation of HEK 293T undergone different experimental treatments (B) The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. (C) The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions. The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.

Function verification of RDF in HEK 293T cell