IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/09
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Working on LovTAP.Reporter systems: PCR to fuse trpL promoter
In order to construct the reporter system regulated by LovTAP, I and Zepeda are working together to fuse the promoter trpL with a reporter gene. As we are interested in characterize LovTAP activator and repressor activity. We have designed the following constructions.
trpL promoter fused to GFP protein:Part:BBa_E0240
Both constructions will be inserted in plasmid pSB3K3 for measurements.
1.Insert the trpL promoter through a PCR reaction to the plasmid pSB1C3.
-trpL promoter sequence
-Primer_trpL_reverse (5'->3'): SpeI site + trpL promoter + XbaI site + EcoRI site GGACTAGTCCCTTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCCTCTAGAAGCGGCCGCGAATTC
-Primer forward (5'->3'): Suffix
-Template: Plasmid pSB1C3
The reaction starts at 95°C during 5 min.
Each cycle is programmed as follows:
After the cycle 35, the temperature changes to 72°C during 5 min and ends at 4°C.
Results:PCR with promoter trpL