Tochingyuet (Talk | contribs) |
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− | Our result showed that, at least one switch for each viral gene worked as expected and expressed increased amount of RFP when its respective trigger is together co-transformed (switches: PB2-3, H7-3, N9-1, N9-2, H5-2, N1-1). In addition, our SAT switch also showed increased amount of RFP when trigger is co-transformed. | + | Our result showed that, at least one switch for each viral gene worked as expected and expressed increased amount of RFP when its respective trigger is together co-transformed (switches: PB2-3, H7-3, N9-1, N9-2, H5-2, N1-1). In addition, our SAT switch also showed increased amount of RFP when trigger is co-transformed. |
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<h4>Cell free system</h4> | <h4>Cell free system</h4> | ||
− | We tried to express: (1) luciferase positive control provided by the manufacturer, (2) negative control where DNA is replaced with water in separate cell free reactions, (3) J61002(constitutive mRFP generator as a positive control of the highest possible RFP expression),(4) N9-2 toehold switch, and (5) N9-2 toehold switch and trigger pair, in cell free system. However, we cannot obtain any significant fluorescent signal from any of the cell free reaction (data not shown). To check if any protein is expressed (M.W. of mRFP= 25.4kDa; M.W. of luciferase= 62.9 kDa), we ran an SDS-PAGE gel: | + | <p style="font-family: roboto;font-size:115%;"> |
+ | Since N9-2 switch showed the highest ON/OFF ratio (33 fold), we further investigate its performance in cell free system. We tried to express: (1) luciferase positive control provided by the manufacturer, (2) negative control where DNA is replaced with water in separate cell free reactions, (3) J61002(constitutive mRFP generator as a positive control of the highest possible RFP expression),(4) N9-2 toehold switch, and (5) N9-2 toehold switch and trigger pair, in cell free system. However, we cannot obtain any significant fluorescent signal from any of the cell free reaction (data not shown). To check if any protein is expressed (M.W. of mRFP= 25.4kDa; M.W. of luciferase= 62.9 kDa), we ran an SDS-PAGE gel: | ||
<center><img src="https://static.igem.org/mediawiki/parts/9/9e/CUHK_cellfreegel.jpeg" width="30%" height="auto"></center> | <center><img src="https://static.igem.org/mediawiki/parts/9/9e/CUHK_cellfreegel.jpeg" width="30%" height="auto"></center> | ||
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We could not observe any over-expression of desired protein in the gel. Possible reason behind is that the protein is not expressed due to nuclease contamination(cell free system is extremely sensitive to nuclease) or is expressed in unobservable quantity. | We could not observe any over-expression of desired protein in the gel. Possible reason behind is that the protein is not expressed due to nuclease contamination(cell free system is extremely sensitive to nuclease) or is expressed in unobservable quantity. | ||
Latest revision as of 19:17, 1 November 2017