Results of the blue light system

Developing The Blue Light Inducible Promoter

This promoter, inspired from the 2014 USTC China iGEM team is a blue light sensitive promoter that expresses gets repressed in the presence of blue light, i.e., greater the intensity of blue light, the more it would get repressed. This means that as we change the intensity of blue light, the expression of the gene downstream of the promoter will change. Here, I have used mrfp as the reporter gene for testing the blue light inducible promoter.

Shown above (Fig. 1) is the final plasmid I will be constructing which will be transformed into E.coli MG1655 cells for experimental characterization of the promoter. Shown below (Fig.2) is the promoter construct which is a composite part consisting of 6 parts out of which 3 are genes, 2 are promoters and one double terminator (Dterm).

The C.P constitutively expresses the YF1 and FixJ genes. YF1 is a chimeric gene made of Ytv1 (which is expressed to produce a light-sensitive protein) and FixL (that phosphorylates FixJ and activates it). The phosphorylated FixJ induces pFixK2 and hence mrfp is expressed. As a result, the mrfp is constitutively expressed when there is no blue light. In the presence of blue light, FixL is unable to phosphorylate FixJ and this ability to phosphorylate reduces with increased intensity of blue light. Unphosphorylated FixJ is unable to induce pFixK2 as a result of which mrfp downstream isn’t fully expressed. In short, increasing the intensity of blue light reduces the expression of gene downstream of pFixK2. With RFP being the reporter protein, I aim to characterize this promoter.

Results

1.1 Transformation and plasmid extraction

I started with some of the first things to be done which was to increase the concentration of some of the plasmids (containing individual genes required for building the gene circuit mentioned above) that we got from the iGEM headquarters as they’re usually low in concentration. So, we transformed the 6 plasmids into E.coli DH5alpha competent cells through heat shock. Competent cells were prepared using the standard Hanahan protocol for making chemically competent cells (Refer protocol section for more details). The plasmids were then isolated using the HiPurA™ Plasmid DNA Miniprep Purification Kit.
I isolated the plasmids containing YF1+FixJ gene, mRFP gene, pFixK2 promoter, Double terminator (Dterm) (the 2 red dots in the circuit diagram) as shown in the table 1 below. The constitutive promoter (C.P) containing plasmid was isolated by another team member.

Table 1: List of bio bricks used in building this promoter

List of Primers used

1.2 PCR amplification and overlapping PCRs

I made use of restriction-free cloning, PCRs, for obtaining the final composite promoter and all other intermediate parts which I will be describing below.
First, I amplified Dterm (using primers dtermSM1_FOR and dtermSM1_REV), pFixK2 (using primers pFixK2_FOR and pFixK2_REV), mrfp (using primers RFPSM1_FOR and suffix_REV) from their respective plasmids. For amplification of parts from plasmids, 50-100 ng of template was used. For Dterm, annealing temperature (A.T) was 53 deg., for pFixK2 the A.T was 58 deg. The extension period (E.P) for both was 18 sec. For mrfp, A.T was 54 deg. and an E.P of 50 sec was used (Fig.3) The resulting bright bands obtained in each case were gel extracted using the Qiagen PCR/Gel cleanup kit (Please refer the protocols section).

Next, I PCR amplified Constitutive promoter (C.P) and the YF1+FixJ construct from their respective plasmids. Here too, 50-100 ng of template was taken for amplification. For C.P, annealing temperature used was 55 degrees with an extension period of 15 sec using primers prefix_FOR and CP_REV. The band of interest was gel extracted and purified (Fig.4). For the YF1+FixJ construct on the other hand, annealing temperature of 54 degrees and an E.P of 2 min (using primers YF1_FOR and YF1_REV) was used which gave 2 bands one of which was the band of interest. This band was gel extracted and purified (Fig.5)

Table 3: The concentrations of parts obtained post gel extractions which will further be used for overlapping PCRs for putting together different parts in the order shown in Fig.2

The first ones to be put together were Dterm and pFixK2 (TP). The two genes were used as template and a 3-step overlap extension PCR along with primers (dtermSM1_FOR and pFixK2_REV) was performed to stitch them together at an A.T of 52.2 deg. and an E.P of 30 sec. About 50 ng of pFixK2 was used and equimolar quantity of Dterm was used. The resultant product was gel extracted and purified (Fig.6).

Now the YF1+FixJ gene construct along with TP were used as templates for Overlap extension PCR for putting them together to make YTP. 31.7 ng of YF1+FixJ construct and equimolar concentration of TP was used. Here, a two-step PCR was used along with primers (YF1_FOR and pFixK2_REV) instead of a 3-step because the annealing temperatures of the primers were very close to the extension temperature of 68 deg. of the GXL polymerase (refer protocols section). An E.P of 2 min 20 sec was used. The obtained product was run on a gel and extracted (Fig.7).

Now, I used pFixK2 and mrfp as my templates to perform extension PCR to make PR. 200 ng of mrfp and equimolar concentration of pFixK2 was used. First, a 3-step PCR with just the templates (without forward and reverse primers) at an A.T of 51 deg. And an E.P of 50 sec for 15 cycles. Now, the forward and reverse primers (pFixK2_FOR and suffix_REV respectively) were added to the PCR mix and the A.T was kept the same whereas the E.P was changed to 3 min for another 15 cycles. The resulting product was gel extracted and purified (Fig.8).

Finally, I used YTP and PR as my templates to perform an overlap extension PCR to obtain the final construct (CYTPR) using 215 ng of YTP and equimolar concentration of PR. How I did this was by first performing a 2 step PCR with just the templates (no primers) as the annealing temperature was higher than the extension temperature (68 deg.) of the polymerase (Because now the entire pFixK2 was an overlapping region). This was done for 15 cycles with an E.P of 2 min. After 15 cycles, forward and reverse primers (CP_FOR and suffix_REV respectively) were added, of which, the forward primer consisted of the entire constitutive promoter. An A.T of 54 deg. And E.P of 3 min 10 sec was used. The product was run on a gel, gel extracted and purified (Fig.9).

Looking at the gel, the concentration seemed very low (as a light band is obtained for 5 uL of product run). So, this product was now used as a template for re-amplification of the final construct. About 50 ng of template was used along with a completely new set of bigger forward and reverse primers (ConstProm_FOR and mrfp_REV). Since these primers are long and have an annealing temperature of 69-70 deg., again a two step PCR was performed with an extension period of 3 min 10 sec. The resulting product was gel extracted and purified. Fig 10 shows that the concentration of the final construct has increased, since now, a much brighter band is obtained with just 2 uL of product.

Now, This insert was digested with Pst1-HF and Xba1 in CutSmart buffer. The pSB1A2 backbone (provided to us in linearized backbones by iGEM 2019) was also digested with the same enzymes for 2 hours at 37C and heat inactivating at 80C for 20 min. The 2 products were ligated using NEB T4 DNA ligase in it's buffer for 8 hours and were directly transformed into DH5 alpha competent cells. The colonies obtained were screened using YF1_FOR and YF1_REV for amplifying YF1-FixJ gene of the insert. As we can see in the colony PCR below (Fig. 11), we have one positive colony against the positive control.

Characterization

The following characterization has been done for the positive colony using the primary inoculation itself. The 3rd colony was inoculated into 3 different cultures- one for dark, one for blue light and the one more for white light. The positive control for this characterization is constitutively expressing RFP which was bacteria containing the plasmid of Const Promoter. 3 different inoculations were done for the positive control as well, one for every light condition. For detailed protocol, please look at Experiments.

As we can see above in Fig.12, we compare across different light conditions, we see that the rfp output is lowest in blue light as expected from the model as well as Literature because in the presence of blue light, the phosphorylation of Yf1 by FixJ is inhibited leading to less induction of pFixK2 and hence lower rfp output . Although we see a higher fluorescence in white light whereas we should have seen the highest output in dark but this opens up opportunities for further investigations of the effect of light of differing wavelengths on the interaction between YF1 and FixJ to induce pFixK2 and hence rfp expression.

We are people of culture too!

Team:IISER-Pune-India/Results/Blue-Light