Difference between revisions of "Part:BBa K2905018"
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[[File:T--Alma--readthrough_reporter1.png|600px]] | [[File:T--Alma--readthrough_reporter1.png|600px]] | ||
| − | This plate clearly shows that the reporter is functional for producing the blue chromoprotein. Visualizing red fluorescence is not as easy, although there seems to be more red hue in MRA8 than in DH5a. Measuring fluorescence is also complicated by the fact that the chromoprotein, cjBlue, and RFP share absorption spectra. | + | This plate clearly shows that the reporter is functional for producing the blue chromoprotein. Visualizing red fluorescence is not as easy, although there seems to be more red hue in MRA8 than in DH5a. Measuring fluorescence is also complicated by the fact that the chromoprotein, cjBlue, and RFP share absorption spectra. The presence of a fused RFP protein, however, can still be detected via SDS PAGE of whole cell lysates. |
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| + | [[File:T--Alma--tag_gel1.png|800px]] | ||
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| + | The above is a representative gel for whole cell lysates (cells grown in LB + 1mM IPTG + Chloramphenicol for 3 hours, pelleted , lysed and loaded onto a 12% SDS PAGE gel) of the three aforementioned E. coli strains containing K2905018. As can be seen, C321.dA leads to a much higher amount of large molecular weight protein than the other strains, as expected. | ||
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Latest revision as of 20:43, 21 October 2019
TAG-Read through reporter
A reporter construct - normally expresses a blue chromoprotein, but will also produce a red protein if the TAG codon does not terminate translation
Measuring fluorescence and expression: Alma 2019
To determine if the part is working correctly, we transformed it into DH5a, MRA8 (A temperature sensitive RF1 strain), and C321.ΔA.exp (a strain in which RF1 has been deleted - C321.ΔA.exp was a gift from George Church; Addgene plasmid # 49018). The expectation is that DH5a should display a blue color, while MRA8 and C321 may show a higher degree of red. C321 grew slowly, but displayed results similar to that of MRA8 - it is not included on the representative plate below.
This plate clearly shows that the reporter is functional for producing the blue chromoprotein. Visualizing red fluorescence is not as easy, although there seems to be more red hue in MRA8 than in DH5a. Measuring fluorescence is also complicated by the fact that the chromoprotein, cjBlue, and RFP share absorption spectra. The presence of a fused RFP protein, however, can still be detected via SDS PAGE of whole cell lysates.
The above is a representative gel for whole cell lysates (cells grown in LB + 1mM IPTG + Chloramphenicol for 3 hours, pelleted , lysed and loaded onto a 12% SDS PAGE gel) of the three aforementioned E. coli strains containing K2905018. As can be seen, C321.dA leads to a much higher amount of large molecular weight protein than the other strains, as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1525
Illegal AgeI site found at 1637 - 1000COMPATIBLE WITH RFC[1000]