Results of R-R System

Overview

R-R (Reporting and Regulation) system consists of three integrated parts, the inclusion body induced red fluorescence reporting system, the inclusion body induced cell lysis regulation system, and the TPA positive feedback regulation system. The first and second systems are applied in E.coli and the third system is applied in Saccharomyces cerevisiae. The red fluorescence was detected by 96-well Microplate Reader or directed observation after centrifugation (12000rpm, 1min). The cell lysis was detected by measuring the OD₆₀₀ of culture medium. The TPA positive feedback effects were determined by the expression level of downstream gene RFP, which was measured by detecting the red fluorescence.

Detailed results

1.Results of inclusion body based reporting system.

The red fluorescence can be observed by bare eyes. We set up four groups:
A. No E.coli.
B. E.coli with empty plasmid pUC19.
C. E.coli with pUC19+CpxR-RFP.
D. E.coli with pUC19+CpxR-RFP+PETase gene.
The result is as the following picture. (After centrifugation with the speed of 12000rpm for 1min)

Fig.1. Directed observed fluorescence of inclusion body based reporting system. (From left to right: A, B, C, D)

2.Results of inclusion body based cell lysis regulation system.

We first did a experiment to measure the cell lysis effect of ddpX gene. We simply use the IPTG inducible T7 promoter to regulate the expression of ddpX gene. We totally set four groups:
1. E.coli wildtype.
2. E.coli wildtype added IPTG.
3. E.coli with ddpX gene, no induction.
4. E.coli with ddpX gene, IPTG added as induction.
Then we continuously measure the OD₆₀₀ of the culture medium by 96-well Microplate Reader and draw the OD₆₀₀-culturing time curve by Matlab. The graph is showed below.

Fig.2. OD₆₀₀-culturing time curve of different groups.

3.Results of TPA positive feedback regulation system.

Summary

In this part of experiment, we have achieved the following results:

1.We successfully applied and improved the previous part BBa_K339007 designed and constructed by the Group iGEM10_Calgary to construct our inclusion body based reporting system and inclusion body based cell lysis regulation system.

2.The red fluorescence can even be directly observed by bare eyes when we express the PETase gene in the E.coli which was transformed into the CpxR-RFP fragment.

3.When we replace the RFP gene with the ddpX gene, when the PETase gene was expressed, we detected the decrease of OD of culture medium, which showed the lysis of E.coli.

4.The TPA positive feedback system can be induced by TPA. We used RFP as reporting gene and found that the higher the TPA concentration is, the stronger the red fluorescence could be detected. We believe that if the RFP gene is replaced by our PETase gene, the expression of PETase gene can also be induced by the PET degradation product TPA.