Part:BBa_K629010
recAp-cheZ-eGFP
Background
recA-cheZ-eGFP.In our project, this part consist of recAp , cheZ, and eGFP when this part is under irradiation, recAp can be started to promote the expression of CheZ, which will make the direction of rotation of the flagellar motor counterclockwise and E. Coli would move forward. Without irradiation or E. Coli moves into a normal environment, the quantity of CheZ would decrease dramatically, leading the direction of rotation of the flagellar motor to be clockwise. As a result, this part will make e.coli move forward to the radiation source. On the other hand, eGFP will express and show the location of E.coli.
Expression level in E.coli
The expression of part CheZ-GFP on plasmid pUC18 in E.coli.
Figure1 chez-GFP-pUC18-control
Figure2 chez-GFP-pUC18-experiment
Because we cannot see fluorescence of recA-GFP-pET28a-BL21 and recA-GFP-pET28a-BL21 in Laser Scanning Confocal Microscope, we turned to Microplate Reader, a machine that can measure the activity of enzyme. We set the exciting wave at 488nm and the emission wave at 520nm, which allow us the measure the fluorescence of recA-GFP-pET28a-BL21 and recA-GFP-pET28a-BL21. After induce with NAL and regulate the OD value of each inocula to a same value, we measured the fluorescence intensity of recA-GFP-pET28a-BL21 and recA-GFP-pET28a-BL21.
Figure3 Fluorescence intensity of recA-GFP-pET28a-BL21 in the first and second experiments
Figure4 Fluorescence intensity of recA-GFP-pET28a-BL21 in the third and forth experiments
Discussion
About the two crests of the curve of recA-GFP-pET28a-BL21, we guess that the rec promoter inside the genome of the BL21 itself, beside the one caused by rec promoter in the plasmid, may be the reason of another crest. At first, the recA promoter is triggered by the increasing NAL concentration, which leads to the first crest. As the NAL concentration increases, the DNA of the plasmid was damaged but the rec promoter in the genome has not been triggered yet since the concentration of the NAL has not reached its threshold, which results in the trough between the two crests. Finally, the rec promoter in the genome is triggered by the relatively high concentration of NAL and produces the protein of rec family, which may help repair the DNA of the plasmid, which causes the second crest of the curve.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| n/a | recAp-cheZ-eGFP |