Team:SVCE CHENNAI/Demonstrate
Wet Lab Results
RNA Thermometer With GFP reporter
ABOUT THIS PART:
The part (BBa_K2379005) consists of RNA thermometer followed by GFP. The RNA thermometer(FourU) (BBa_K115002) is a temperature sensitive element that shows higher expression of fluorescence at 37ºC and lower expression below 37ºC. The part works as expected and maximum level of fluorescence was observed at 37ºC.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes).
- Charechterization studies was carried out at 32 degrees celcius, 37 and 42. The cultures at 42 degrees celcius were gown at 37 until 0D600 of 0.3 was attained.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600, background fluorescence was minimized using a negative control.
- Triplets were used for characterisation and the error bars indicate the Standard deviation of their mean values.
CONCLUSION:
The expression of GFP was found to be basal and minimum at 32ºC. A sudden induction in the expression was observed at the temperature 37ºC and it seemed to be gradually decreasing after 42ºC. This behaviour of RNA thermometer(FourU) observed supports the data given by other teams. Thus, the RNA thermometer(FourU) works as expected.
Adaptor With RNA Thermometer
ABOUT THIS PART:
This part (BBa_K2379010) is a variant of the adaptor (BBa_K2379008) . We used it to regulate gene expression at 37ºC. RNA thermometer(FourU) (BBa_K115002) is a temperature sensitive element used in this part that shows higher expression at 37ºC and lower expression below 37ºC. The part works as expected. When GFP is placed downstream of this gene, maximum fluorescence was observed at 37ºC.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes).
- Charechterization studies was carried out at 32 degrees celcius, 37 and 42. The cultures at 42 degrees celcius were gown at 37 until 0D600 of 0.3 was attained.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600, background fluorescence was minimized using a negative control.
- Triplets were used for characterisation and the error bars indicate the Standard deviation of their mean values.
CONCLUSION:
Similar results were obtained for this part containing RNA thermometer with adaptor as obtained for RNA Thermometer (FourU) + GFP except for the fact that in this case RNA thermometer(FourU) is able to control and regulate the transcription of downstream genes with the help of adaptor element which was not possible earlier.
How can we infer that the adaptor is working?
The adaptor is working because it controlled the expression of GFP through the RNA thermometer, which is only possible had the RNA thermometer regulated thermosensitive expression at the transcriptional level and not the translational level as GFP has its own constitutive RBS. Had the adaptor not worked, even at 32 degrees celcius the construct would have given high levels of expression. To give a comparison we also charechterized the adaptor with a constitutive RBS, its studies is below.
Adaptor With Constitutive RBS
ABOUT THIS PART:
This part (BBa_K2379009) is a variant of adaptor which is regulated by constitutive RBS (BBa_B0034) . Since it contains the constitutive RBS, it shows similar levels of fluorescence expression irrespective of temperatures. The part works as expected and when GFP is placed downstream of this gene, similar levels of fluorescence was observed at 32ºC, 37ºC and 42ºC.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes).
- The cell cultures were grown initially at temperature 37ºC until it reached an OD600 of 0.3. Then the cultures were transferred and incubated at three different temperatures: 32ºC, 37ºC and 42ºC.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600, background fluoresence was minimized using a negative control.
- Triplets were used for characterisation and the error bars indicate the Standard deviation of their mean values.
CONCLUSION:
The part works as expected. Since there is no temperature based regulatory element, similar fluorescence was observed at all the three different temperatures irrespective of the time points.
RNA pHmeter with Wild Scar
ABOUT THIS PART:
This part (BBa_K2379006) functions as a pH-sensitive riboswitch and we used it to regulate gene expression at high pH (above 7.5). It consists of the natural 4bp scar site. The part works as expected i.e. when GFP is placed downstream of it, maximum fluorescence was observed at pH 8.5.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes)
- The cell cultures were grown at three different pH : 7.0 ,8.0 and 8.5. Fluorescence values were recorded at excitation and emission peaks of 460nm and 515nm respectively.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600.
- Duplets were used for characterisation and the error bars indicate the Standard deviation of the mean values.
CONCLUSION:
Expected results were obtained for the RNA pHmeter (Wild scar) while using GFP as the reporter gene. There was only an average 20.5% increase in the expression of GFP from pH 7 to pH 8.5 at all the three time points .Though this was not the case in the native operon where the expression was influenced largely by the SraF gene, the discrepancy in the values are the due to the influence of both the pLac promoter and the SraF gene on the expression of GFP. Yet the SraF gene holds it’s control over gene expression which is evident from the maximum fluorescence peak observed at pH 8.5. This serves as a proof of concept for working of RNA pHmeter with wild scar.
RNA pHmeter with Biobrick scar
ABOUT THIS PART:
This part (BBa_K2379007) functions as a pH-sensitive riboswitch and we used it to regulate gene expression at high pH (above 7.5). It consists of the natural 4bp scar site. The part works as expected i.e. when GFP is placed downstream of it, maximum fluorescence was observed at pH 8.5.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes)
- The cell cultures were grown at three different pH : 7.0 ,8.0 and 8.5. Fluorescence values were recorded at excitation and emission peaks of 460nm and 515nm respectively.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600.
- Duplets were used for characterisation and the error bars indicate the Standard deviation of the mean values.
CONCLUSION:
Expected results were obtained for the RNA pHmeter containing biobrick scar site. There was only an average 22.06% increase in the expression of GFP from pH 7 to pH 8.5 at all the three time pointswhich is comparable to that obtained for RNA pHmeter with wild scar. This proves that the changes made in the gene sequence hasn’t affected the way in which the part functions. Though the expression here is controlled by both the promoter and the SraF gene, the latter has more ascendancy which is evident from the maximum fluorescence observed at pH 8.5 irrespective of the time points at which it is recorded. Thus the RNA pHmeter with biobrick scar works as expected.
RNA pHmeter Adaptor
ABOUT THIS PART:
This part is used to convert translational regulation of the RNA pHmeter ( BBa_K2379002 ) to transcriptional regulation by means of the adaptor ( BBa_K2379008 ) .The part works as expected. When GFP is placed downstream of the pLac promoter, transcription and translation of downstream genes were induced at pH 8.5.
CHARACTERISATION STUDIES FOR THIS PART:
- The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes)
- The cell cultures were grown at three different pH : 7.0 ,8.0 and 8.5. Fluorescence values were recorded at excitation and emission peaks of 460nm and 515nm respectively.
- Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600.
- Duplets were used for characterisation and the error bars indicate the Standard deviation of the mean values.
CONCLUSION:
The results obtained for the part having adaptor combined with RNA pHmeter is as expected. There was an average 25.46% increase in the expression of GFP from pH 7 to pH 8.5 at all the three time points which is certainly higher than that obtained for RNA pHmeter without adaptor. We can infer that the adaptor is working as it regulated the expression of GFP in a pH sensitve manner which is only possible had there been regulatory control at the transcriptional level, as GFP has its own constitutive RBS and translatational level would have resulted in constitutive expression of GFP.
REFERENCES
- Konan, K.V. & Yanofsky, C. Rho-Dependent transcription termination in the tna Operon of Escherichia coli: Roles of the boxA Sequence and the rut Site.J. Bacteriol. 182(14), 3981–3988 (2000).
- Chang, C. L.,Lei Qi, Lucks J.B., Segall-Shapiro T.H., Wang, D.,Mutalik V.K.& Arkin, A.P.An adaptor from translational to transcriptional control enables predictable assembly of complex regulation. Nat. Methods 9,1088–1094(2012).
- Richardson J.P. Loading Rho to terminate transcription. Cell 114, 157–159 (2003).
- Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A.,Westhof, E., & Altuvia, S. A pH-responsive riboregulator.Genes Dev. 23, 2650–2662 (2009).
- Nechooshtan, G., Elgrably-Weiss, M., & Altuvia, S.Changes in transcriptional pausing modify the folding dynamics of the pH-responsive RNA element. Nucleic Acids research. 42(1), 622-630 (2013)
- Bingham, R., Karen, H., Joan S. Alkaline induction of a Novel Gene Locus, alx, in Escherichia coli. Journal of Bacteriology. 172(4), 2184-2186(1990)
