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From 2013.igem.org

Contents 1 Background 2 Our System 3 Methods 4 Results

Background

Recently, techniques have been developed that afford researchers the ability to control gene expression with light. To date, sensors that respond to red, green, and blue wavelengths of light have been reported. Light induced expression of genes has a number of advantages over chemical induction methods.  For example, light induced expression is cheaper than chemical methods, can be used to finely tune expression levels through modulations of intensity, and can be rapidly removed -- a feature lacking in chemical induction systems. Furthermore, many light expression systems are reversible, depending on the wavelength of light used.  Finally, the ability to control the expression levels of multiple genes of interest simultaneously could have far reaching implications for tuning biosynthetic pathways. The use of chemical inducers for this application would likely prove to be difficult and cost prohibitive, but multichromatic gene induction represents a potential solution to this problem. To this end, the 2013 University of Washington iGEM team chose to continue a project we began in 2012 that is aimed at the development and characterization of a set of tools that bring multichromatic gene expression into the realm of possibility for synthetic biologists.

In 2012, the University of Washington iGEM Team developed a freely available app for the Android operating system that affords spatiotemporal control over light wavelengths and intensities. When installed on an appropriate tablet (see E.Colight) the app could be useful for carrying out complex light induced expressions. In 2012, we were unable to fully characterize the app’s ability to control expression of light inducible genes. Thus, the 2013 iGEM Team’s goals include fully characterizing the app’s ability to control gene expression with light, and the development of a toolkit of light induced expression biobricks that are confirmed to work with the tablet app.

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Our System

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The light induced expression systems we used both require multiple protein components. In general, a small molecule chromophore is synthesized in the cell, and acts as a substrate for a transmembrane light sensing protein. When the protein sensor bound-chromophore-bound is exposed to light of the correct wavelength, a conformational change is induced in the sensor protein which in turn causes the phosphorylation of a transcription factor. The active form of the transcription factor then modulates the expression of any gene downstream of its cognate promoter.  We worked with a red and green light inducible gene expression system this year.  Both systems utilize the same chromophore, phycocyanobilin, but differ in the transmembrane light sensing proteins that bind the chromophore, and the downstream regulator proteins. 

Source	Component	Function
Chromophore:	Plac/ara-HO1-PcyA	PCB chromophore expression plasmid
pPLPCB	Plac/ara	Hybrid lactose and arabinose promoter
pPLPCB	HO1	Heme oxygenase
pPLPCB	PcyA	Phycocyanobillin ferridoxin oxidoreductase
Green light sensor:	[PCpcG2-RBS-sfGFP-CcaS](rev)-CcaR	Sense green light with reporter output
pJT119b	CcaS	Green light receptor
pJT119b	CcaR	Response regulator
pJT119b	PcpcG2	Synechocystis phycocyanoybillin promoter
pJT119b	BBa_B0034	Strong Elowitz RBS
pJT119b	sfGFP	Superfolder GFP reporter
Red light sensor:		Sense red light with reporter output
pEO100c	POmpC-BBa_B0034-sfGFP	Red light inactivated GFP output
pEO100c	POmpC	OmpC promoter
pEO100c	BBa_B0034	Strong Elowitz RBS
pEO100c	sfGFP	Superfolder GFP reporter
pJT106b	PLambda-RBS-LacZ-T1-POmpC-CI-T1	Red light activated GFP output
pJT106b	Plambda-CI-Term	Output inverter

The genes required to construct a functional light induced protein expression system were generously provided by Jeff Tabor at Rice University. Table 1.1 lists the genes required for each system and their respective functions. In order to construct a functional light responsive system, a multiple genes are transformed into E. coli.  Regardless of the system in question, the minimal components required are: 1) genes utilized in the synthesis of the chromophore, 2) a transmembrane light sensing protein, and 3) response regulators which are activated by from the light sensitive proteins, and ultimately function as transcription factors.

Our red and green light sensitive systems were constructed through transformation of a subset of the plasmids listed in Table 1 into BL21 E. coli cells. Our green light responsive system was constructed by transforming pPLPCB and pJT119b simultaneously. A red light responsive system was created by transforming pPLPCB, pCPH8, and pEO100c into E.coli. GFP was used as the output of both systems.

Table 1: Description of source plasmids and components they contain

Methods

Cloning

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Each light-responsive biosensor is composed of two or three plasmids, which requires three vectors with compatible origins of replication and orthogonal antibiotics. To subclone the light responsive operons into Biobricks, we chose equivalent copy number and antibiotic iGEM vectors. Because tetracycline is more common in iGEM vectors than spectinomycin, we chose pSB3T5 to put the chromophore operon into.

Source Vector	Origin	Copy Number	Antibiotic	Biobrick Equivalent	Antibiotic	Biobrick Name
pJT119b	ColE1,pMB1	100-300	Chloramphenicol	pSB1C3	Chloramphenicol	pBB_GonG
pPLPCB	p15A	20-30	Spectinomycin	pSB3T5	Tetracycline	pBB_PCB
pEO100c	pSC101	~5	Ampicillin	pSB4A5	Ampicillin	pBB_RoffG
pJT106b	pSC101	~5	Ampicillin	pSB4A5	Ampicillin	pBB_RonL

Plasmids were transformed into either XL1-blue or DH5a cells and DNA was harvested after overnight growth using a miniprep kit from Qiagen. Genes of interest were amplified using oligos with Prefix and Suffix added on to the ends, plus 3-6 extra basepairs recommended by NEB for efficient cutting. Operons from pPLPCB, pJT119b and pEO100c were amplified using oligos that compatible with insertion into the pSB1C3 backbone between Prefix and the Suffix; pJT106b was also put into pSB4A5.

Light Methods

This year our team developed a protocol optimized for the specifics of our system. Overnight cultures were grown of E. coli containing plasmids with genes for all of the parts of the light system. The next morning, these cultures were used to inoculate either M9 media or M9 agar. The app was set up for the appropriate container and light conditions, and the “dark” plate is covered in aluminum foil. These plates are placed on the tablet and grown in a 37C incubator. Cell density (absorbance) and GFP output (fluorescence) are then measured using a plate reader. We ran multiple experiments using this general protocol, including a timecourse, intensity test, and blinking test, plate image test. Timecourse experiment was done in varying amount of time points. For our plasmid, we tested them up to 16 hours. Intensity experiment was also run for our green light inducible GFP. Different percentage of intensity which could be selected in the tablet. Blinking test incorporates different timing between green and dark which could also be specified using our app. Finally we inoculate cells into media which also contains agar. Then the media + agar plates are subjected to different light conditions.

Results

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