Team:IIT Bombay India/Analysis

Experimental Studies

Objective

1. Characterize the expression profile of synthetic network designed with multiple feedback loops.

2. Characterize beta-galactosidase expression in the mutant strains of E.coli lacking lacI containing the designed synthetic genetic circuits.

3. Phenotypic characterization of the transformed strains by quantifying growth on lactose

Plasmid Developed

1: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K255001 BBa_K255001] - pLac.laci-cfp.pTet.yfp, cloned in pSB1A2.

2: [http://partsregistry.org/Part:BBa_K255002 BBa_K255002] - pTet.laci-cfp.pTet.yfp, cloned in pSB1A2.

3: [http://partsregistry.org/Part:BBa_K255002 BBa_K255003] - pLac.laci-cfp.pTet.yfp, cloned in pSB2K3.

4: [http://partsregistry.org/Part:BBa_K255002 BBa_K255004] - pTet.laci-cfp.pTet.yfp, cloned in pSB2K3.

Above plasmids have been deposited in repository of standard biological parts, iGEM-2009

Strains Developed

Ecoli(MR1655,lacI deleted) strain was transformed individually with each plasmids by chemical methods.

The strains were selected on the basis of their resistance to growth on Ampicillin and kanamycin. This resistance was provided to them by the respective plasmid which they have incorporated.

Strain 1 (Open Loop) with plasmid (BBa_K255004). It has got open loop without any feedback. Here there is constitutive expression of lacI. Here the copy number of the plasmid is fixed.

Strain 2 (Single Input Single Output with regulation on LacI [SISO_LacI]) with plasmid (BBa_K255003). It has got a single negative feedback loop. So the expression of lacI is under regulation. Here also the copy number of the plasmid is fixed.

Strain 3 (Single Input Single Output with regulation on copy number [SISO_CN]) with plasmid(BBa_K255002). It has got single negative feedback loop on the plasmid copy number . Here there is no control on the LacI expression.

Strain 4 (Multiple Input Multiple Output with regulation on copy number and LacI [MIMO]) with plasmid (BBa_K255001). It has dual negative feedback loop one on the plasmid copy number and second on the LacI expression.

Experimental study

1. Characterization of copy number by quantifying YFP for all the four strains developed.

2. Characterization of YFP to see the effect of temperature on the copy number of plasmid.

3. Growth studies on lactose for host strain (lacI deletion) and host strain transformed with synthetic genetic circuits having multiple feedback loops.

4. Characterization of beta-galactosidase expression from host strain ( lacI deletion) and host strain transformed with synthetic genetic circuits having multiple feedback loops.

Experimental Protocols

Here is a detailed link to experimental protocol.

Results

1. The YFP expression in Strain-1 (open loop) and strain 2 (SISO_LacI) was found to be nearly same at 0, 100, 200 and 500 micrometre of IPTG concentration. YFP expression for strain-3 (SISO_CN) increased by 10 fold on increasing IPTG concentration from 0 to 100 micrometre of IPTG. Thereafter it remains nearly constant. The variability in the distribution of YFP as characterized by FACS demonstrated that Strain-4 had the minimum variability in protein expression indicating lower noise.

2. At low temperature (30 degree Celsius) the plasmid copy number decreased for all the strains but at 42 degree Celsius the plasmid copy number decreased for Strain 3 and Strain 4 but we could not find any difference in the copy number for strain 1 and strain 2 from 37 degree Celsius.

3. The growth rate on lactose of Strain-1 was lower as compared to Strain-4. Further, the growth of Strain-4 was more sensitive to lower concentration than that observed in Strain-1. The variability in the growth rate was lower for strain-4 indicating that the multiple feedback loop yields robust protein expression which translates to stable growth rates. Agar plate experiments also demonstrated similar results.

4. The beta-galactosidase expression of strain-4 was commensurate to the lactose concentration demonstrating that the multiple feedback yields optimal behavior. Strain-1 demonstrated lower beta-galactosidase activity due to higher LacI in the system. This added burden in strain-1 (open loop) reduced the growth rate.

Conclusion

In our iGEM -2009 project we have experimentally demonstrated the effect of multiple feedback loops on the gene expression using synthetic genetic circuits with none, one and two negative feedback loops. Experiments demonstrated that the system with multiple feedback loops was less noisy compared to others. The copy number was quantified by measuring YFP expression using FACS to obtain the mean expression and variability... The experimental studies demonstrated that the variability observed in the protein expression was propagated to the phenotypic level. Both agar plate experiments and growth rate measurements demonstrated lower variability for the strain with multiple feedback loops as compared to the open system. The growth experiment also demonstrated that the strain with multiple feedback loop design could optimally synthesize proteins to the availability of lactose thus minimizing burden of protein synthesis and maximizing growth rate.

Complete Experimental results can be seen here.