Team:UANL Mty-Mexico/Wet lab/Light experiments

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:

1. Testing the function of our light inducing device (Light Machine) by using original Dr. Jeff Tabor's plasmids.
2. 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.

Also, another experiments were planed to test our new parts. Each of them are detailed in the next paragraphs.

Preparing light-inducible E. coli JT2 cells.

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

Testing Light Machine

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

Integration procedure
Steps	Status
Transforming with helper plasmid pTKRED	Done
Integration of Landing Pad	Done
Transforming with pTKIP plasmid.	Done
Recombination of pTKIP plasmid with Landind Pad.	In progress

1. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res 38:e92.