Difference between revisions of "Part:BBa K352001:Experience"
(→Culturing CO Sensor E.coli with CO induction) |
(→Applications of BBa_K352001) |
||
| Line 34: | Line 34: | ||
===Applications of BBa_K352001=== | ===Applications of BBa_K352001=== | ||
| + | <br>We hope to provide a technical guideline for other future gas sensing cell sensor projects with our E-CO sensor with enhanced dynamic range project. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 15:13, 28 October 2010
Electrophoretic Mobility Shift Assay (EMSA)
Result:
As indicated, slight retardation in PCOOF and PCOOM bands are observed. On the other hand, it is difficult to conclude an affinity difference between these promoters based on the intensity of retarded bands.
Intrinsic Tryptophan Fluorescence (ITF)
Result:
pink:reduced - CO bounded CooA + buffer
yellow : reduced –CO bounded CooA + 28 nM pCooF ( 20. Min)
blue: reduced –CO bounded CooA +56 nM pCooF ( 40. Min) </br>
green: NC: reduced –CO bounded CooA + 10 ul elution buffer ( 20.min)
purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer (40. Min )
As shown in the graph, there is an decrease in the intensity of the fluorescence; however when looked at negative control measurements, we saw the decrease in intensity because of the dilution and time effects. So we cannot say that the reduction is caused from pCooF – CooA interaction. Moreover, we suspect that heme group in the CooA quench fluorescence of the tryptophan and also heme group gives absorbance at 280- 400 nm so our protein might give the fluorescence at that range itself.
Applications of BBa_K352001
We hope to provide a technical guideline for other future gas sensing cell sensor projects with our E-CO sensor with enhanced dynamic range project.
User Reviews
Culturing CO Sensor E.coli with CO induction
We have used 300 mL flasks with 80 mL LB culture. We have applied 80 Ml Carbon monoxide to some. The results were successful. Cultures induced with carbon monoxide successfully expressed red fluorescent protein.
There is a bit red color in flask without any CO exposure. This might be because cooA protein is transcriptional activator not a transcriptional factor. Therefore, a leakage have been observed in our experiments; however, there is very good differentiation which is sensible.
Reference: Aono S (2003). Biochemical and Biophysical Properties of the CO-Sensing Transcriptional Activator CooA. Accounts of Chemical Research 2003; 36(11): 825-831.
UNIQ2be6cf48f19c58ff-partinfo-00000006-QINU UNIQ2be6cf48f19c58ff-partinfo-00000007-QINU