Overview
Based on BBa K3244013 and BBa K3755010, we added a new part BBa K4421031(improved NFAT response element), which was random mutanted from BBa K3755010, but with a much higher response strength.
Improvement background
In our project, we designed a kill-switch circuit which involved NFAT response element.
NFAT serves as a block signal, which can be ignited by antigen stimulation. When the stimulation occurs and the concentration of NFAT meets the requirements, Gal4-KRAB should be produced quickly to block the promoter of the suicide gene, inhibiting the suicide process. So we require our NFAT response element to be of high response strength.
We searched in the registry for existing part.
BBa_K3244013 designed by iGEM19_AFCM-Egypt and BBa_K3755010 designed by iGEM21_ShanghaiTech_China were available for us to improve.
1.In Part: BBa_K3244013, they generally introduced the biological background of NFAT response element, but with no experiment results.
2.In Part: BBa_K3755010, they cotransfected NFAT-RE+minP+EGFP(Part:BBa K3755014) and Piezo1.1 (BBa K3755006, calcium signal provider) into HEK 293 cells and through fluorescence colocalization, they observed some cells had produced EGFP successfully, which meant NFAT and NFAT response element functioned well.
Design
We performed 33 random mutations on BBa_K3755010.
Note: iGEM21_ShanghaiTech_China replaced the luciferase in pGL4.30 with EGFP for lack of luciferin; while we took the original pGL4.30 to carry out the experiments.
Result
Taking BBa_K3755010 as control, from the figure, we can clearly see that the response strength of mutation 13 is significantly higher than the control and other mutants, promising to achieve ideal results in our experiment.
>BBa_K3755010
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
>mutation 13 (26: g→c)
ggaggaaaaa ctgtttcata cagaacgcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt