Part:BBa_K360121:Experience
Characterizing LovTAP
Once LovTAP with the three weak constitutive promoters (J23117,123114 and J23105) and the strong promoter (J23102) was correctly obtained in plasmid pSB3K3, and the reporter system trpL+RFP was also finished in plasmid pSB1C3 , the co-transformation procedure was done, in order to have the whole system inside E.coli cells, both trpR wildtype and mutant. Besides, trpL+RFP construction in plasmid pSB1A2 from Lausanne team was kindly sent it by Edinburgh team. Both trpL-RFP reporter systems were used, being the Lausanne system the reference, expecting to obtain similar results. The difference between the reporter systems is that the constructed in plasmid pSB1C3 doesn’t have the transcriptional double terminator.
In order to test if LovTAP works correctly two protocols were implemented: the qualitative and the quantitative approach.
Considerations to take into account:
- The RFP protein does not include a degradation tag, so the time required to notice a clear difference between light and dark states will be long, because although LovTAP starts to repress, the RFP produced previously by the cells will be still present.
- The plasmid in which LovTAP is being express is pSB3K3 with a copy number around 20 to 30, while trpL-RFP construction is inside plasmids pSB1C3 and pSB1A2; both are high copy number plasmids (100 to 300 per cell). So there are many trpL binding sites that should be repressed by LovTAP. The ideal condition for this experiment would be to have LovTAP and trpL+RFP constructions inside the same plasmid.
- Using the LovTAP constructions fused to promoters with different strength, it can be tested at what levels of expression, the LovTAP light regulation is better. It is already known that under high expression levels of LovTAP, trpL promoter is repressed even in dark. To test this scenario J23102 promoter was used.
Qualitative experiment
The qualitative approach was designed to observe and compare the RFP production in the cells harboring LovTAP exposed to light versus dark conditions, both in wild type and trpR mutant strains. Under blue light conditions, when LovTAP repressor activity is activated, it is expected to observe a lower level of RFP in comparison to the cells maintained in the dark state.
Experimental procedure
The samples used were: trpL+RFP reporter system in plasmid pSB1C3, Lausanne trpL-RFP reporter system, LovTAP under promoters J23117, J23114, J23105 and J23102.
Only one colony of each co-transformation was used along the experiment and was tested under light and dark conditions.
TrpR mutant and wild type cells were co transformed using 5 µL of each plasmid (trpL-RFP in pSB1C3/ pSB1A2 and each LovTAP construction in pSB3K3). For each co transformation one tube containing 5 ml of LB medium with Kanamicyn and chloramphenicol or Kanamicyn and ampicillin, was inoculated from a single colony of DH5α. Cultures were grown overnight (~15hrs) at 37°C with spinning at 250rpm in dark conditions. Then, 1 ml of broth was taken and transferred into 5 ml of fresh LB medium with antibiotics. The cultures were grown for approximately 13 hours under the previous conditions but some were exposed to blue light (470nm) while others were maintained in dark. Finally, the cultures were spun down and compared as follows: the RFP pellets obtained under blue-light versus dark condition, and wild type samples versus mutant samples.
Results
Only one colony of each co-transformation was used along the experiment and was tested under light and dark conditions.
In a first approach was observed that wt and trpR mutant cells co-transformed with LovTAP under J23102 promoter grew in dark and light, produced very low levels of RFP protein in comparison with the control sample (wild type cells with only the reporter construction, trpL+RFP). Besides, there was no a visible difference between dark and light conditions, which is the expected behavior under high expression levels of LovTAP. Considering this result we decided to continue the co-transformations procedure without considering the LovTAP construction under J23102 promoter.
Using LovTAP with promoters J23105, J23114 and J23117 (in order of high to lower strength), we obtained the following pellets that are show in the next images:
According to the images trpR mutants seem to have a lower RFP expression levels versus WT both in dark and light conditions. This is a surprising result because it was expected that showed higher levels than WT as they don’t have the possible crass-talk with trpR native E.coli repressor. Maybe there is another process of trpL repression independent to LovTAP and trpR.
Both trpL-RFP reporter systems give similar results. Comparing blue-light versus dark exposed samples there seems to be a small difference visible by naked-eye, with higher levels of RFP protein in dark state samples. Maybe the small difference observed between light and dark conditions is because LovTAP protein levels are very low to considerably repress the trpL promoter. As well, the long half-life of the RFP protein could be masking a significant difference between both states.
Although these results not formally demonstrate that the LovTAP repression is light dependent, it seems that in cells co-transformed with LovTAP versus those transformed only with the trpL-RFP system, there are lower levels of RFP. Another interesting pattern observed in the samples, is that there are lower levels of RFP as the promoter strength that regulates LovTAP increases, thus suggesting that there is a gradient of repression depending on the LovTAP concentration.
These results suggest that possibly LovTAP is working well, however as this experiment is totally qualitative, it must be improved taking into account the optical density of the samples, because the observations could be due to the presence of different number of cells in each sample. As well replicates of the experiment are needed.
Quantitative experiment
With the aim to have a better characterization of LovTAP, we designed a new protocol considering the methodology describe by Jason R Kelly, we include some changes that are detailed below.
We decided to start the protocol under blue light conditions to test the dark state, expecting that in dark exposed samples the RFP levels increase in comparison with those samples that are always under blue light. We did this in order to face better the issue of the long half life of the RFP due to it doesn’t have a degradation tag.
Experimental procedure
The samples used were: trpL+RFP reporter system in plasmid pSB1C3, Lausanne trpL-RFP reporter system, LovTAP under promoters J23117, J23114, J23105 and RFP under J23102.
Only one colony of each co-transformation was used along the experiment and was tested under light and dark conditions.
TrpR mutant and wild type cells were co transformed using 5 µL of each plasmid (trpL-RFP in pSB1C3/ pSB1A2 and each LovTAP construction in pSB3K3). As controls RFP constitutively expressed under J23102 promoter was used and wild type cells transformed just with trpL-RFP. For each transformation one tube containing 5 ml of LB medium with Kanamicyn and chloramphenicol or Kanamicyn and ampicillin, was inoculated from a single colony of DH5α. Cultures were grown overnight (~14hrs) at 37°C with spinning at 100rpm under blue light (470nm) conditions. Then cultures were diluted 1:1000 into 5ml of fresh LB media with antibiotics. These were grown for approximately 4.5 hours under the previous conditions under blue light (470nm). After this step the OD600 was measured from each culture. Based on this OD measurement, the cultures were diluted to the same OD (0.15) in 5 ml of LB fresh media. Then three 200 µL aliquots from each culture were transferred into a flat-bottomed 96 well plate. Samples were loaded in the plate as is shown in the next picture:
An initial measurement of OD600 and fluorescence (530/25 nm excitation filter, 590/35 nm emission filter) by triplicate was done. Using the light emission device to irradiate the incubator, the plate was maintained during 17 hrs at 37°C with spinning at 35rpm inside the incubator but with a mask in the samples selected for the dark conditions. Then, a second measurement of OD600 and fluorescence was done. Initial data with the second measurement were compared. Background absorbance and fluorescence were determined by measuring wells containing only media, and the values obtained were used to normalize the other samples.
Results
The average ODs of the wells with only media and antibiotics changed drastically from the initial to the second measurement(km/amp from 0.282 to 0.09, and km/cm 0. to 0.093), dificulting the data analysis. So the data obtained were inconsistent and even it was intented to analyse them, there was not the expected difference between light and dark conditions.
Conclusions
According to the aforementioned results, we think that LovTAP could possibly work well. However we have to improve the characterization protocols in order to support with enough evidence this conclusion. As well, more replicates and controls must be included in the experiments.
We also think that using other reporter system to describe the transcriptional activation activity of LovTAP using a cascade of double repression, might generate better results. So we will test LovTAP behavior with the cI inverter.
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