Part:BBa_K3251001
IPTG-induced LuxR expression with chromoprotein
This part includes an addition of a second chromoprotein, BBa_E1010, to the biobrick BBa_K3251000. This red reporter protein serves to visualize the level of 3OC6HSL. In the presence of IPTG, the LuxR gene encodes for the LuxR protein and the yellow chromoprotein, which serves as a control. Binding of the LuxR+3OC6HSL complex will activate the luxpR promoter, which allows the expression of the red chromoprotein. Depending on the concentration of 3OC6HSL, the combination of the red and the yellow chromoproteins will be visualized with a color gradient ranging from yellow to red.
Characterization by team GCGS_China 2021
We conducted an induction experiment to qualitatively test the part.This part includes an addition of a second chromoprotein, BBa_E1010, to the biobrick BBa_K3251000 .
Qualitative expression experiment
Firstly, we have the part synthesized by the company with the pUC57 plasmid as backbone, then we transformed it into component cell E.Coli DH5α.
1. Inoculation
Adding 5mL LB and 5μL AMP respectively to three shake tubes, a, b, c, then these tubes were overturned for 20 times.
Picking single clone of E.coli DH5α that contained synthesized plasimd from the culture
medium by using pipette tips. The culture picked was added to the tubes a, b, c respectively.
The tubes were placed in the shaker and shook for 8-12 hours in 37℃
2. Adding IPTG to induce the amiGFP.
Adding 5mL LB, 5μLAMP and 5μL, 0.1M IPTG to three shake tubes D, E, F respectively.
50μL bacteria liquid from a, b, c were added to D, E, F respetively.
The tubes were placed in the shaker and shook for 12 hours in 37℃
Observing the color of the bacteria cultures overnight.
3. Adding C6-HSL to induce mRFP protein
Adding 5mL LB, 5μL AMP, 5μL IPTG （0.1M）and 50μL C6-HSL(0.01 mM) to the shake tubes D, E, F respectively.
50μL bacteria liquid from a, b, c were added to A, B, C respectively
The tubes were placed in the shaker and shook for 12 hours in 37℃. And it was also clear that the protein was expressed as we expected, for there was no color change of the bacteria that could be observed.
Results
It was clear the protein did not express as we expected by our step by step induction experiments, we didi not observe any color change.
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 2328
Illegal AgeI site found at 2440 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1710
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