Difference between revisions of "Part:BBa K575026"
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(<small>--[[User:ishayardi|ishayardi]] 20:24, 16 October 2018 (UTC)</small>) | (<small>--[[User:ishayardi|ishayardi]] 20:24, 16 October 2018 (UTC)</small>) | ||
| − | ==Team RMHS_Maryland 2018: Characterization of AI-1 Induced Fluorescence in | + | ==Team RMHS_Maryland 2018: Characterization of AI-1 Induced Fluorescence in <i>Escherichia coli</i> for use in co-culture== |
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<img src="https://static.igem.org/mediawiki/2018/7/72/T--RMHS_Maryland--bl21graph.png" width=750><br><br> | <img src="https://static.igem.org/mediawiki/2018/7/72/T--RMHS_Maryland--bl21graph.png" width=750><br><br> | ||
| − | <img src="https://static.igem.org/mediawiki/2018/2/2d/T--RMHS_Maryland--bl21table.png" width= | + | <img src="https://static.igem.org/mediawiki/2018/2/2d/T--RMHS_Maryland--bl21table.png" width=400 height=250><br><br> |
<img src="https://static.igem.org/mediawiki/2018/1/1a/T--RMHS_Maryland--bl21pics1.png"><br><br> | <img src="https://static.igem.org/mediawiki/2018/1/1a/T--RMHS_Maryland--bl21pics1.png"><br><br> | ||
<img src="https://static.igem.org/mediawiki/2018/2/24/T--RMHS_Maryland--bl21pics2.png"><br><br> | <img src="https://static.igem.org/mediawiki/2018/2/24/T--RMHS_Maryland--bl21pics2.png"><br><br> | ||
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<i>RESULTS</i><br> | <i>RESULTS</i><br> | ||
| − | Team RMHS_Maryland 2018 obtained characterization data for this construct in a BL21 E. coli chassis (BL21 is a strain that produces a constant high level of AI-2). Overnight cultures of BL21 transformed with BBa_K575026 on the standard backbone were grown and diluted 50x in LB media. AI-1 (3-oxo-C12-HSL) was added and fluorescent microscope images were taken at time intervals 1.5, 3 and 4 hours after initial incubation with AI-1. Percent of cells fluorescing was determined by dividing the cell count under the fluorescent microscope with the cell count under brightfield.<br><br> | + | Team RMHS_Maryland 2018 obtained characterization data for this construct in a BL21 <i>E. coli</i> chassis (BL21 is a strain that produces a constant high level of AI-2). Overnight cultures of BL21 transformed with BBa_K575026 on the standard backbone were grown and diluted 50x in LB media. AI-1 (3-oxo-C12-HSL) was added and fluorescent microscope images were taken at time intervals 1.5, 3 and 4 hours after initial incubation with AI-1. Percent of cells fluorescing was determined by dividing the cell count under the fluorescent microscope with the cell count under brightfield.<br><br> |
Although it may be clearly observed that fluorescence is relatively minimal even when AI-1 is added, the data indicates that the promoter is, as in part BBa_K575024, not prone to leaky expression, and that RFP expression does in fact occur. The percent fluorescence increased a great deal between 3 and 4 hours after incubation, but even at 1.5 and 3 hours it is evident from the microscope images that there were at least a few cells fluorescing, indicating that, although it would likely not serve as a very good reporter, this part does indeed work as specified. | Although it may be clearly observed that fluorescence is relatively minimal even when AI-1 is added, the data indicates that the promoter is, as in part BBa_K575024, not prone to leaky expression, and that RFP expression does in fact occur. The percent fluorescence increased a great deal between 3 and 4 hours after incubation, but even at 1.5 and 3 hours it is evident from the microscope images that there were at least a few cells fluorescing, indicating that, although it would likely not serve as a very good reporter, this part does indeed work as specified. | ||
</html> | </html> | ||
Revision as of 01:30, 17 October 2018
LasR/PAI1 Inducible Promoter + RBS (B0030) + RFP, Constitutive Promoter + RBS (B0030) + LasR
This construct contains a LasR/PaI1 (3-oxo-C12-HSL) inducible promoter with an RBS and an RFP reporter. There is also constitutive transcription of the LasR receptor protein. Thus, the promoter will activate and fluorescence will be observed in the presence of PAI1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 58
Illegal NheI site found at 907
Illegal NheI site found at 930 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1286
Illegal AgeI site found at 741
Illegal AgeI site found at 853
Illegal AgeI site found at 1483 - 1000COMPATIBLE WITH RFC[1000]
[http://2018.igem.org/Team:RMHS_Maryland Team RMHS_Maryland 2018] contributed to the characterization of this part by demonstrating that the part produces RFP expression when induced by AI-1.
(--ishayardi 20:24, 16 October 2018 (UTC))
Team RMHS_Maryland 2018: Characterization of AI-1 Induced Fluorescence in Escherichia coli for use in co-culture
RESULTS
Team RMHS_Maryland 2018 obtained characterization data for this construct in a BL21 E. coli chassis (BL21 is a strain that produces a constant high level of AI-2). Overnight cultures of BL21 transformed with BBa_K575026 on the standard backbone were grown and diluted 50x in LB media. AI-1 (3-oxo-C12-HSL) was added and fluorescent microscope images were taken at time intervals 1.5, 3 and 4 hours after initial incubation with AI-1. Percent of cells fluorescing was determined by dividing the cell count under the fluorescent microscope with the cell count under brightfield.
Although it may be clearly observed that fluorescence is relatively minimal even when AI-1 is added, the data indicates that the promoter is, as in part BBa_K575024, not prone to leaky expression, and that RFP expression does in fact occur. The percent fluorescence increased a great deal between 3 and 4 hours after incubation, but even at 1.5 and 3 hours it is evident from the microscope images that there were at least a few cells fluorescing, indicating that, although it would likely not serve as a very good reporter, this part does indeed work as specified.