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Characterising LovTAP

Once LovTAP with the three weak constitutive promoters (J23117,123114 and J23105) and strong promoter (J23102) was correctly obtained in plasmid psb3k3, and the reporter system trpL+RFP was also finished by our team in plasmid psb1c3 , we started the co-transformation procedure in order to have the whole system inside the cells both trpR wild type and trpR mutant. Besides, we also receive the trpL+RFP construction in plasmid pSB1A2 from Lausanne team that was kindly sent it by Edinburgh team, because we didn’t get any response from the members of Lausanne team to provide us with their reporter systems. We are using both our trpL-RFP reporter system and the Lausanne system as a reference, expecting to obtain the similar results. The difference between the reporter systems is that ours doesn’t have the double terminator.

In order to test if LovTAP works correctly we implemented two protocols: the qualitative and the quantitative approach. With the qualitative approach we only want to observe and compare the RFP production in the cells exposed to light vs dark conditions, both in wild type and trpR mutant strains. What we are analyzing is the repressor activity of LovTAP, thus under light condition- when LovTAP repressor activity is activated, we expect to observe a lower level of RFP in comparison to the cells maintained in the dark state.

Considerations to take into account:

• The RFP protein doesn’t include a degradation tag so the time required to observe 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, 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, we can test at what levels of expression, the LovTAP light regulation is better. We already know that under high expression levels of LovTAP, trpL promoter is repressed even in dark. To test this scenario we used J23102 promoter.

Qualitative experiment

Experimental procedure

1.Co-transform the cells using 5 microliters of each plasmid in the trpR mutant and in the wild type strains. The samples used are:

• Our trpL+RFP reporter system plus:

J23102+LovTAP. J23117+LovTAP. J23114+LovTAP. J23105+LovTAP.

• Lausanne trpL+RFP reporter system plus:

J23117+LovTAP. J23114+LovTAP. J23105+LovTAP.

2.Grow up the transformed cells overnight (~15hrs) in 5ml of LB medium at 37°C with spinning at 250rpm, with the respective antibiotics (Kanamicyn and chloramphenicol/ Kanamicyn and ampicillin ) in dark conditions. 3. Take 1 mL of broth and transfer it into 5 ml of fresh LB medium with antibiotics. 4. Incubate the cells in the two different conditions: the blue light and dark states for ~ 13 hrs.

Blue-Light samples were kept in the incubator covered in aluminium foil with 4 leds inside. The samples in dark conditions were maintained in the non-light devices that were constructed with bottles and newspaper.

5. Spin down the cultures and compare the RFP pellets obtained under blue-light vs. dark condition, and wild type samples vs. 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 we 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). So, it seems that we are observing the expected behavior under high expression levels of LovTAP. However we couldn’t notice a difference between dark and light conditions.

With this previous 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:

• Samples with our trpL+RFP reporter.

Samples description is detailed here:

Rows listed from top to bottom and Columns from left to right.

Row, Column. LovTAP promoter, Strain phenotype, Condition

1,1.J23105,trpR mutant,Dark

1,2.J23105,Wild type,Dark

1,3.J23105,trpR mutant,Blue Light

1,4.J23105,Wild type,Blue Light

2,1.J23114,trpR mutant,Dark

2,2.J23114,Wild type,Dark

2,3.J23114,trpR mutant,Blue Light

2,4.J23114,Wild type,Blue Light

3,1.J23117,trpR mutant,Dark

3,2.J23117,Wild type,Dark

3,3.J23117,trpR mutant,Blue Light

3,4.J23117,Wild type,Blue Light

• Samples with Lausanne trpL+RFP reporter.

Samples description is detailed here:

Rows listed from top to bottom and Columns from left to right.

Row, Column. LovTAP promoter, Strain phenotype, Condition

1,1.J23105,trpR mutant,Dark

1,2.J23105,Wild type,Dark

1,3.J23105,trpR mutant,Blue Light

1,4.J23105,Wild type,Blue Light

2,1.J23114,trpR mutant,Dark

2,2.J23114,Wild type,Dark

2,3.J23114,trpR mutant,Blue Light

2,4.J23114,Wild type,Blue Light

3,1.J23117,trpR mutant,Dark

3,2.J23117,Wild type,Dark

3,3.J23117,trpR mutant,Blue Light

3,4.J23117,Wild type,Blue Light

According to the images trpR mutants seem to have a lower RFP expression levels vs WT both in dark and light conditions. This a surprising result because we 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 that we are not taking into account independent to LovTAP and trpR.

Both trpL-RFP reporter systems give similar results. Comparing blue-light vs 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 demostrate 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 we must improve it taking into account the optical density of the samples, because our observations could be due to the presence of different number of cells in each sample.

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 in the article “Measuring the activity of Bio-brick promoters using an in vivo reference standard”, 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.

Only one colony of each co-transformation was used along the experiment and was tested under light and dark conditions.

1.Co-transform the cells using 5 micro liters of each plasmid in the trpR mutant and in the wild type strains.

J23117/ 123114 / J23105 LovTAP + our trpL+RFP reporter system. J23117/ 123114 / J23105 LovTAP + Lausanne trpL+RFP reporter system.

2.Grow up the transformed cells for ~20 hrs in 5ml of LB medium at 37°C with spinning at 250rpm, with the respective antibiotics (Kanamicyn and chloramphenicol/ Kanamicyn and ampicillin ) in Blue-light conditions.

3. Dilute the cultures to 1:100 into 5ml of fresh LB media with antibiotics.

4. Grow the cells for 4 hours at 37°C with spinning at 250rpm in Blue-light conditions.

5. Measure the optical density OD600 of 500 microliters of each culture and dilute them to an ODE of 0.15.

6. Transfer three 200 microliters aliquots from each culture into a flat-bottomed 96 well plate.

Samples were loaded in the plate as is shown in the next picture:

7. Make an initial measurement of OD and RLU of the plate.

8. Using the light emission device constructed to illuminate each well of the plate with one led. Keep the plate at room temperature during 10 hrs under the desired light and dark conditions.

9. Make a second measurement of OD and RLU of the plate.

10. Compare initial data with the second measurement.

Results

We had problems during the development of the protocol in the steps of OD measurements, and unfourtunately the initial data before the treatment of the plate with light and dark conditions were unsaved. So we couldn't compare the differences before and after the light and dark exposition of the samples in the plate.

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.

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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