Difference between revisions of "Part:BBa K1732015"

Revision as of 23:26, 18 September 2015

J23115-B0034-T7RNAP-LBD-B0034-YFP

T7 RNA polymerase with a constitutive promoter. YFP is added to the end of the plasmid so that the amount of T7 RNA polymerase that is produced can be quantified through fluorescence analysis of YFP (iGEM14_Carnegie_Mellon).

The graph above demonstrates that J23100 promoter allows for expression of the RFP compared to the J23115 promoter by approximately 8 fold. This is expected because the J23100 promoter is a stronger promoter, allowing for more RFP to be produced.

Although the T7 RNAP is expressed using a weaker constitutive promoter (J23115), it results in the expression of higher levels of fluorescence compared to that the constitutively expressed J23100 promoter, indicating its ability to produce high levels of protein. Thus, if the goal was to synthesis large concentrations of luciferase protein, it would be more efficient to use the T7 system.

Construction

In order to test reporters and BEAM, the team's estrogen sensor from last year ([http://2014.igem.org/Team:Carnegie_Mellon/Our_Sensor 2014 Estrogen Sensor]) was improved. The biosensor is a bacterial cell containing two-plasmids. The sensor plasmid is a high-copy plasmid which has the ligand binding domain of the human estrogen receptor alpha (ER-LBD) inserted into T7 RNA polymerase (T7 RNAP) and YFP for normalization. When the ER-LBD binds estrogen, it causes a conformational change (ref) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (ref). T7 RNAP is a strong phage RNA polymerase that requires no additional factors. The second plasmid that makes up our sensor is a low-copy plasmid, the reporter plasmid, which has the T7 promoter driving expression of RFP. When the T7 RNAP is reconstituted upon binding to estrogen, it allows for binding to the T7 promoter on the reporter plasmid and transcription of the RFP mRNA which then is translated to produce RFP.

Improvements

Last year's biosensor used an intein which had 3 components: the N-terminus of the S. cerevisiae VMA intein, the human estrogen receptor ligand binding domain, and the C-terminus of the intein all inserted into T7 RNAP between amino acids 491 and 492. We were unable to get any significant red fluorescent signal from our sensor cells in the presence of estrogen last year. This version of the sensor which does not use an intein and was positioned between residues 179 and 180 of T7 RNAP and was able to give us significant fluorescent signal in the presence of estrogen. The sensor is now functional and successfully detects estrogen whereas the previous version did not.

The reporter plasmid was then modified to express Gaussia luciferase.

Experiments with Estrogen Biosensor using Gaussia Luciferase Modified Reporter Plasmid

Experiments were performed to demonstrate whether the addition of beta-estradiol to the biosensor using the Gaussia luciferase modified reporter plasmid had an effect of the luminescence levels in the presence of 10 ul of 2 uM coelenterazine. Overnight cultures of the biosensor in MACH cells were grown, one with beta-estradiol (estrogen +) and one without (estrogen -). A control of J23100 RFP was used as a control because no luminescence was expected to be expressed even with the addition of coelenterazine. The biosensor in the presence of beta-estradiol showed a 5 fold increase in luciferase activity.

Controls

For these experiments there were three controls that did not contain the ER-LBD. The first control was intact T7 RNAP with no YFP and the third control had YFP (BBa_K1491034). The second control had restriction sites in place of the ER-LBD (BBa_K1732016). The sites added the amino acids ACLKLGGSTGGGSHNC between K179 and K180.

Experiments

The improved sensor and controls were tested using a variety of growth protocols to evaluate the response to estrogen. A TECAN plate reader was used to measure red and yellow fluorescence after overnight exposure to various concentrations of 17-beta-estradiol. The controls showed no response and the sensor cells showed differences in RFP signal ratioed to YFP signal at concentrations ranging from 1nM to 100 uM.

Experiments were performed to test the protocol for the estrogen sensor and the sensitivity of the sensor. The most reliable sensor protocol was using overnights from single colonies which were then restarted; this gave us consistent starting cells for the assay. There were also 3 controls that were tested as well. One control had no YFP. The second control had restriction sites in place of the estrogen receptor ligand binding domain. The third control had YFP and no restriction sites. The sensor showed a three-fold or more increase in mRFP fluorescence signal upon addition of estrogen while the controls showed relatively no increase in mRFP fluorescence signal. Concentration data was also acquired. Concentrations of 100 uM, 20 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, and 0 nM beta-estradiol had their fluorescence tested. As expected, the more beta-estradiol present, the higher the mRFP signal acquired.

Conclusions

The overnight/restart data with controls proves that, unlike the previous version of the sensor, this version functions as desired and clearly indicates that estrogen is present. The concentration data proves that the sensor system is able to indicate relative amounts of estrogen present as well.

The ER LBD appears to be behaving within T7 RNAP as it does within split Venus (Shis and Bennet 2012). However, we observed a bimodal response to estrogen, either a 2 fold response or a 7-10 fold response, the source of the variation is uncertain.

Sources of Sequence

The position within T7 RNAP was from Shis DL and Bennet MR. 2012. Library of synthetic transcriptional AND gatesbuilt with split T7 RNA polymerase mutants. PNAS 110: 5028-5033.

The ER LBD was from McLachlan MJ, Katzenellenbogen JA, Zhao H. 2011. A new fluorescence complementationbiosensor for detection of estrogenic compounds. Biotechnol Bioeng 108(12):2794-803

Applications

Our improved part can now be used as a sensor to detect the presence of estrogen. Further testing could include testing the sensor with estrogenic compounds other than beta-estradiol.

References

Routledge EJ, Sumpter JP. 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241–248.

Sequence and Features