Difference between revisions of "Part:BBa K1913016"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <p> This part contains an operon where mRFP (BBa_K081014) is expressed under a modified lambda cI promoter (BBa_I12006). This promoter is (according to the parts page) induced by lambda cI and repressed by 434 cI. This last operon also includes a LVA-tagged lambda cI (BBa_K081007) encoding gene, in theory establishing positive feedback of the modified lambda cI promoter.</p> | ||
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<p>We have combined BBa_K1913016 and BBa_K1913007 into BBa_K1913006, inserted an RBS library (see design notes) upstream of the 434 cI gene and transformed it to DH5alpha. DH5α cells transformed with the BBa_K1913006 plasmid yielded clones with varying intensities of red coloring on agar (without both L-Arabinose and D-Glucose) plates (Figure 1). The variation in the red intensity is seen very clearly between the top three circled colonies in Figure 12.This diversity is an indication that BBa_K1913016 works as intended: variation in the 434 cI RBS should cause different ratios between λ and 434 cI protein levels, leading to differences in mRFP expression. </p> | <p>We have combined BBa_K1913016 and BBa_K1913007 into BBa_K1913006, inserted an RBS library (see design notes) upstream of the 434 cI gene and transformed it to DH5alpha. DH5α cells transformed with the BBa_K1913006 plasmid yielded clones with varying intensities of red coloring on agar (without both L-Arabinose and D-Glucose) plates (Figure 1). The variation in the red intensity is seen very clearly between the top three circled colonies in Figure 12.This diversity is an indication that BBa_K1913016 works as intended: variation in the 434 cI RBS should cause different ratios between λ and 434 cI protein levels, leading to differences in mRFP expression. </p> | ||
Revision as of 11:48, 19 October 2016
434- and lambda cI balance RFP reporter
434- and lambda cI balance RFP reporter
Usage and Biology
This part contains an operon where mRFP (BBa_K081014) is expressed under a modified lambda cI promoter (BBa_I12006). This promoter is (according to the parts page) induced by lambda cI and repressed by 434 cI. This last operon also includes a LVA-tagged lambda cI (BBa_K081007) encoding gene, in theory establishing positive feedback of the modified lambda cI promoter.
We have combined BBa_K1913016 and BBa_K1913007 into BBa_K1913006, inserted an RBS library (see design notes) upstream of the 434 cI gene and transformed it to DH5alpha. DH5α cells transformed with the BBa_K1913006 plasmid yielded clones with varying intensities of red coloring on agar (without both L-Arabinose and D-Glucose) plates (Figure 1). The variation in the red intensity is seen very clearly between the top three circled colonies in Figure 12.This diversity is an indication that BBa_K1913016 works as intended: variation in the 434 cI RBS should cause different ratios between λ and 434 cI protein levels, leading to differences in mRFP expression.
Figure 11. Close-up picture of culture plate with colonies containing the subpopulation two-operon plasmid (including the RBS library). The blue circled and (faintly) red circled colonies were selected to be used in separate plate reader experiments.
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 1470
Illegal AgeI site found at 1582 - 1000COMPATIBLE WITH RFC[1000]