Difference between revisions of "Part:BBa K1732018"

Revision as of 00:23, 19 September 2015

pSB3K3-T7prom-B0034-Gaussia-T7term

In order to test reporters and BEAM (Carnegie Mellon University's DIY fluorimeter and luminometer), the team's estrogen sensor from last year (link to last year's wiki) 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.

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 second control had YFP. The third control had restriction sites in place of the ER-LBD. The sites added the amino acids ACLKLGGSTGGGSHNC between K179 and K180.

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.

The reporter plasmid was then modified to express Gaussia luciferase.

Sequence and Features