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Team:UANL Mty-Mexico/Wet lab/Light experiments
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Revision as of 03:26, 29 September 2011
Light experiments were planed to demonstrate the function of HuBac community, controlling expression of five different products through a simple light-based code. This experiments mainly consisted as follows:
- Testing the function of our light inducing device (Light Machine) by using original Dr. Jeff Tabor's plasmids.
- 2.- Trials of the HuBac community function using "the simplest version of the circuit" which was designed to demonstrate that biphasic switch works as expected.
For testing that Light Machine worked as expected, light-inducible JT2 cells were transformed with different plasmids as specified in the next table:
| Cell | Description | Plasmids | Constructed |
| Green Cell | Responds to green-light producing LacZ | pJT122, pPLPCB(S) | Done |
| Green Cell (-) | Does not respond to green light due to absence of chromophore. Used as negative control for Green Cell. | pJT122 | Done |
| Red Cell | Responds to red-light producing LacZ | pCph8, pJT106, pPLPCB(S) | Done | Red Cell (-) | Does not respond to green light due to absence of chromophore. Used as negative control for Red Cell. | pCph8, pJT106 | Done |
| RedGreen Cell | Responds to red OR green light producing LacZ. | pJT122, pCph8, pPLPCB(S) | ------ |
| RedGreen Cell (-) | Does not respond to green light due to absence of chromophore. Used as negative control for RedGreen Cell. | pJT122, pCph8 | Done |
Characterization of multiple plasmids transformed into E. coli JT2 strain
Green Cells were plated on LB agar media and incubated overnight in absence of light. After that, plates were exposed to green light (532 nm) during 6 hours. Individual colonies were picked up, resuspended on 5 uL of H20 by vortex. After this,10 uL of X-gal were added (20 mg/mL), and there was an incubation period of 30 minutes at 37ºC, finally samples were centrifuged for precipitating cells.
Negative control used in this case was a duplicate culture of Green Cells that was all-time covered for avoiding light induction.
It may be observed a qualitative difference between sample and negative control.
First test of Light Machine. A) Sample exposed to green light, B) Negative control
Characterization of multiple plasmids transformed into E. coli JT2 strain
