In order to characterize the performance of our recombinase-based biological relays, the following experiments are designed.
●The construction of experimental plasmids
●The composition and principle of the experimental systems
●Genotype testing
●IPTG-concentration-gradient induction
●Cytotoxicity measurement
●The normalization of experimental data
To see the whole name of abbreviation, see lab notebook:
https://2019.igem.org/Team:GENAS_China/Notebook
The construction of experimental plasmids
The left side is the basic plasmid, and the right side is the experimental plasmid derived from the basic plasmid. The building process uses Gibson Assembly or Goldengate assembly.
Six composite parts: [attB-BsaI>-<BsaI-Terminator-attP] are inserted:
phiC31-ECK120034435 （IC35）
TG1-L3S3P22 （IT22）
INT5-ECK120010855 （IF55）
INT7-ECK120033737 （IS37）
INT8-ECK120030221 (IE21）
INT10-ECK120020525 （IT25）
Six basic parts of recombinase coding sequence are inserted:
phiC31：pSET152
TG1：pKU462
INT5：pCis2+7+8+5
INT7：pCis2+7+8+5
INT8：pCis2+7+8+5
INT10：pCis7+10+8
The composition and principle of the experimental systems
If recombinant reaction occurs, sfGFP reporter gene, which is downstream of the recombinant site, will be activated. After SFGFP gene was activated, the results could be observed qualitatively under blue light directly, and the proportion of fluorescent cells and fluorescence intensity could be accurately measured by flow cytometry.
Genotype testing
Specific primers are used to amplify the domain near the recombination site, and the recombination reaction can be determined according to the difference in the length of the product. The product can be sequenced to determine the sequence after recombination. We use this method to test the orthogonality of the recombinase system.
IPTG-concentration-gradient induction
The increase in IPTG-induced concentration will cause a higher expression of recombinase, which will give a greater amount of responded recognition sites. The increase in responded sites will result in a higher expression of GFP. And in our experiment, we equalize the expression rate of GFP and the recombinase rate. Therefore, there is a relationship between the concentration of IPTG and the recombinase rate of the system. On the basis of this correlation, we carried out experiment for measuring the response curve of recombinase systems. We set up IPTG concentration gradient which will cause different expression rate of GFP. Then, by using flow cytometry, we can get the percentage of fluorescent cells, which indicates the recombinase rate (expression rate of GFP), under different IPTG concentration. In this way, we get a set of (IPTG concentration, recombinase rate) data pairs. Also, based on the model we built up (see model https://2019.igem.org/Team:GENAS_China/Modell), we can input our data pair into the model and obtain a best fitting line for each recombinase system, which is the response curve of the corresponding recombinase system. Once the response curve of recombinase is obtained, we can compare it with the response curve of the promotor to get the matching promotor for the induced-expression circuit of this system.
Gradient induction experiments of the experimental system are conducted in 96-well plate. Experimental procedures may vary depending on the purpose, as described in the Measurement page. The flow cytometry data are analyzed by software to obtain the proportion of fluorescent cells and the average value of fluorescence.
Cytotoxicity measurement:
After the 20 hours incubation in the IPTG-concentration-gradient induction and before sampling, the OD600 of each well was measured by a microplate reader.
We can compare the measurement result of the sample with the results of other groups. The difference between the readings of OD600 of each group could be a reference of toxicity characteristic.
The normalization of experimental data
The left of figure 5 is the function of we determine for Ptac-GFP. The middle graph is the [IPTG] verses the recombination rate that we determine in the inducing experiment using steady state measurement. In data analyzing, in order to normalize the data and connect the input of promotor and the output of recombinase, we substitute the y -axis of the [IPTG] verses [GFP] into the x-axis of the function of [IPTG] verses recombination rate measured by steady state measurement and get a function of [GFP] verse recombination rate. By doing so, we are able to normalize the output of promoter ([GFP]) and the output of the recombinase (rate of fluorescence). This method is applied when we use the model to fit and parameterize the experimental data.
