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

This image shows the simplest mechanism by which our circuit can operate. It exemplifies biphasic switch operation can change from one state to another with the stimulation of a single input, in this case the red light.

Circuit Simplest Version

Design

Testing and characterization for composite K566004 and K566006 in E.coli JT2 strain under different light intensities and irradiation times of 30 minutes at 532 nm.

Experimental

G (+) = E.coli JT2 containing pJT122, pPLPCB(S), K566004 (CFP), K566006 (Green light genetic inversor)

G (-) = E.coli JT2 containing pJT122, K566004(CFP), K566006 (Green light genetic inversor)

A light irradiation experiment was carried out in JT2 cultures under two controlled environments, one exposed to light and the other in complete darkness. The samples G (+) and G (-) were incubated overnight at 37°C and then 200uL of the samples were inoculated by triplicate for each intensity.

Both samples were measured in a Fluorometer BioTek Synergy, using special filters for CFP detection and measurement (Exitation filter 420nm ±50 and Emsion filter 460 ±40) before being irradiated to observe an initial fluorescence. After been introduced to the Light Machine, where the exposure time started with 30 minutes of Far-red light(710 nm) to reset the photoreceptors that could be switched during the night at the end of this period the fluorescence was measured. Then began to expose the samples to green light (532 nm) for 2 hours and every 30 minutes a portion of the sample is taken to measure the fluorescence gained or lost.

Results

At the end of two hours the following data was obtained:

Plate Control (-)
**Always in the dark

** It is noted that there is the presence of CFP, over time tends to increase.**

Plate 1 **Irradiated

Conclusions

During this experiment it was demonstrated that the new part K566004 which contains a new Cyan Fluorescent Protein controlled by Mnt regulable promoter works as expected, emitting fluorescence and being excited at the reported wavelength.

This new CFP was optimized for better expression in E. coli based on use of preferential codons, therefore, comparation tests against other CFP would be ideal.

Also, during this experiment it was observed that synthetic construction for CFP protein, may be regulated using light specific wavelengths. Demonstrating capacity of the system to be light-regulated.

*** It is observed that the samples G (+) and G (-) had a great expression of CFP during the Far-red light irradiation and after starting the green light exposure the presence of CFP began to decrease.

For measurements of the samples in the Fluorometer, LB medium was removed (it tends to absorb wavelengths near the ultraviolet range, which can interfere with CFP measurements) and then resuspended in sterile MiliQ water, the sample then was treated with liquid nitrogen to lyse the cells without using chemicals or buffers that could interfiere in the fluorescence measurements.