Tochingyuet (Talk | contribs) |
Tochingyuet (Talk | contribs) |
||
Line 34: | Line 34: | ||
<p><h3>Toehold Switches</h3></p> | <p><h3>Toehold Switches</h3></p> | ||
<h4>Co-transformation</h4> | <h4>Co-transformation</h4> | ||
− | Switch plasmid is transformed either with its respective trigger or empty plasmid into E. coli BL21 (DE3) cells. Single colony of each set-up was picked to grow overnight culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation when log phase was reached. After harvesting the cells and determining the OD600, cells were washed with PBS buffer, concentrated 10x and then aliquoted in 96-well plates to determine the fluorescent signal by exciting at 584nm and measuring at 607nm. Final fluorescent signal were normalized by OD600 to give the final relative fluorescent unit (R.F.U). Below listed the result of our influenza switches and SAT switches: | + | Switch plasmid is transformed either with its respective trigger or empty plasmid into E. coli BL21 (DE3) cells. Single colony of each set-up was picked to grow overnight culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation when log phase was reached. After harvesting the cells and determining the OD600, cells were washed with PBS buffer, concentrated 10x and then aliquoted in 96-well plates to determine the fluorescent signal by exciting at 584nm and measuring at 607nm. Final fluorescent signal were normalized by OD600 to give the final relative fluorescent unit (R.F.U). Below listed the result of our 15 influenza switches and SAT switches: |
<br> | <br> | ||
<br> | <br> | ||
Line 45: | Line 45: | ||
<br> | <br> | ||
Our result showed that, at least one switch for each viral gene 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. Since N9-2 switch showed the highest ON/OFF ratio (33 fold), we further investigate its performance in cell free system. | Our result showed that, at least one switch for each viral gene 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. Since N9-2 switch showed the highest ON/OFF ratio (33 fold), we further investigate its performance in cell free system. | ||
− | + | <br> | |
− | + | <br> | |
+ | <h4>Cell free system</h4> | ||
+ | <br> | ||
<p><h3>Cloning Tools of Toehold Switches and Triggers</h3></p> | <p><h3>Cloning Tools of Toehold Switches and Triggers</h3></p> | ||
Restriction mapping by XhoI and PstI showed correct insertion of our cloning tool (~1000bp) into respective backbones (Switch: pSB4C5(~3200bp), trigger: pSB1K3(~2200bp)). Eco31I could also digest the switch and trigger cloning tool. Below shows the 1% agarose gel: | Restriction mapping by XhoI and PstI showed correct insertion of our cloning tool (~1000bp) into respective backbones (Switch: pSB4C5(~3200bp), trigger: pSB1K3(~2200bp)). Eco31I could also digest the switch and trigger cloning tool. Below shows the 1% agarose gel: | ||
− | + | <br> | |
− | + | <center><img src="https://static.igem.org/mediawiki/2017/8/86/CUHK_SDGel.jpg" width="50%" height="auto"></center> | |
− | + | <br> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
We tested our toehold switch cloning tool when constructing our SAT switch and trigger. We ordered a pair of 60nt oligoes when constructing each switch-expressing plasmid and trigger-expressing plasmid. The complementary oligoes were mixed to give dsDNA insert with sticky end. The dsDNA was then ligated to Eco31I digested plasmid. Ligation product was transformed to E. coli DH5a competent cells. Below showed the plate of E. coli cell transformed with SAT switch-oligoes-ligation product and SAT trigger-oligoes-ligation product viewed under blue light: | We tested our toehold switch cloning tool when constructing our SAT switch and trigger. We ordered a pair of 60nt oligoes when constructing each switch-expressing plasmid and trigger-expressing plasmid. The complementary oligoes were mixed to give dsDNA insert with sticky end. The dsDNA was then ligated to Eco31I digested plasmid. Ligation product was transformed to E. coli DH5a competent cells. Below showed the plate of E. coli cell transformed with SAT switch-oligoes-ligation product and SAT trigger-oligoes-ligation product viewed under blue light: | ||
− | + | <br> | |
− | + | <center><img src="https://static.igem.org/mediawiki/2017/7/72/CUHK_toolresult.jpg" width="50%" height="auto"></center> | |
− | + | <br> | |
− | + | ||
− | + | ||
− | + | ||
We can observed that there were fluorescent colonies and non- fluorescent colonies on each plate. The black arrows point to 3 non- fluorescent colonies. Since fluorescent colonies are probably resulted by self-ligated plasmid, we picked the non- fluorescent colonies to sequence. Sequencing result showed that the switch and trigger were successfully inserted. This proved that our tools worked as expected and provide a convenient way for construction of switch-expressing plasmid and trigger-expressing plasmid. | We can observed that there were fluorescent colonies and non- fluorescent colonies on each plate. The black arrows point to 3 non- fluorescent colonies. Since fluorescent colonies are probably resulted by self-ligated plasmid, we picked the non- fluorescent colonies to sequence. Sequencing result showed that the switch and trigger were successfully inserted. This proved that our tools worked as expected and provide a convenient way for construction of switch-expressing plasmid and trigger-expressing plasmid. | ||
− | |||
− | |||
− | |||
− | |||
− | |||
<p><h3>Characterization of chromoproteins</h3> </p> | <p><h3>Characterization of chromoproteins</h3> </p> |
Revision as of 09:19, 29 October 2017