Difference between revisions of "Part:BBa K3269001"

Latest revision as of 00:34, 22 October 2019

uvrA GFP UV Damage Sensor
UV damage detector
The uvrA promoter involved in Nucleotide Excision Repair (NER) in E. coli cells allows for SOS controlled expression of sfGFP, allowing for a visual output of the amount uvrA is expressed, and therefore the amount of Damage accrued within the genome.

In order to validate that our part performs its intended function of providing a visual fluorescent output that is proportional to the UV dose given to the cell, we exposed E. coli K-12 cells transformed with our part (insert part number here) in a pSB1A3 backbone with our UV-C handlamp at 11.9 W/m^2 for increments of 0, 4, and 10 seconds (setup pictured in Figure 4). Negative (BBa_K3269003) and positive (BBa_K3269002) controls for fluorescence were also tested. Cells were inoculated in LB with ampicillin the night before and then diluted to an OD of 0.1 in M9 media the day of the experiment, and were then allowed to grow up to ~0.4. Directly after exposure in the 96-well plate, cells were then placed in a plate reader, which measured their fluorescence over a 3 hour period, taking Fluorescence and OD600 measurements every 3 minutes. Controls for our part were also exposed under the same conditions.

Our results suggest that there is no significant difference in induction at any of the UV doses for our part BBa_K3269001 This is most likely because we only exposed it to UV transiently as opposed to continuously. Possible improvements could include the addition of an Anderson promoter to recruit more transcription machinery, as well as a stronger RBS to raise levels of protein expression.

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Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 586
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 976
Illegal BsaI site found at 1154

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 The uvrA promoter involved in Nucleotide Excision Repair (NER) in E. coli cells allows for SOS controlled expression of sfGFP, allowing for a visual output of the amount uvrA is expressed, and therefore the amount of Damage accrued within the genome.
-In order to validate that our part performs its intended function of providing a visual fluorescent output that is in some way proportional to the UV dose given to the cell, we exposed E. coli K-12 cells transformed with our part in a pSB1A3 backbone with our UV-C handlamp at 11.9 W/m<sup>2</sup> for increments 0, 4, and 10 seconds. Negative and positive controls for fluorescence were also clones, the positive having a constitutive promoter upstream of GFP (BBa_K3269002) and the negative only having the uvrA promoter and no GFP(BBa_K3269003). Cells were inoculated in LB with ampicillin the night before and then diluted to an OD of 0.1, and were then allowed to grow up to ~0.4. Directly after exposure in the 96-well plate, cells were then placed in a plate reader, which measured their fluorescence over a 3 hour period, taking Fluorescence and OD600 measurements every 3 minutes.
+In order to validate that our part performs its intended function of providing a visual fluorescent output that is proportional to the UV dose given to the cell, we exposed E. coli K-12 cells transformed with our part (insert part number here) in a pSB1A3 backbone with our UV-C handlamp at 11.9 W/m^2 for increments of 0, 4, and 10 seconds (setup pictured in Figure 4). Negative (BBa_K3269003) and positive (BBa_K3269002) controls for fluorescence were also tested. Cells were inoculated in LB with ampicillin the night before and then diluted to an OD of 0.1 in M9 media the day of the experiment, and were then allowed to grow up to ~0.4. Directly after exposure in the 96-well plate, cells were then placed in a plate reader, which measured their fluorescence over a 3 hour period, taking Fluorescence and OD600 measurements every 3 minutes. Controls for our part were also exposed under the same conditions.
 <br>
 <br>
-Sadly, our results suggest that there is no significant difference in induction at any of the UV doses for our part BBa_K3269001. This is most likely because we only exposed it to UV transiently as opposed to continuously. Possible improvements could include the addition of an Anderson promoter to recruit more transcription machinery, as well as a stronger RBS to raise levels of protein expression.
+Our results suggest that there is no significant difference in induction at any of the UV doses for our part BBa_K3269001 This is most likely because we only exposed it to UV transiently as opposed to continuously. Possible improvements could include the addition of an Anderson promoter to recruit more transcription machinery, as well as a stronger RBS to raise levels of protein expression.
 "https://2019.igem.org/wiki/images/2/28/T--Northwestern--UvrAFluorescence.png"