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Team:Illinois/MicF
From 2009.igem.org
MicF Target-GFP Fusion
Purpose: MicF will be fused to the target sequence from the OmpF gene to the GFP gene on a low-copy plasmid and transform this into E. coli cells along with a high-copy plasmid carrying the MicF gene. We expect to see translational repression of GFP by the small RNA MicF.
Protocol(s) Used: (links to protocols page)
Recipe(s) Used:
Primers Used: (5' -> 3')
- Forward MicF (sRNA gene) primer: 5'p+GCTATCATCATTAACTTTATTTATTACCG
- Reverse MicF (sRNA gene) primer: GTTTTTTCTAGAGGTAGCACAGAATAATGAAA
- Forward OmpF (sRNA gene) primer: GTTTTTATGCATAGACACATAAAGACACCAAACTC
- Reverse OmpF (sRNA gene) primer: GTTTTTGCTAGCAGGGACGATCACTG
MicF gene sequence:
- GCT ATC ATC ATT AAC TTT ATT TAT TAC CGT CAT TCA TTT CTG AAT GTC TGT TTA CCC CTA TTT CAA CCG GAT GCC TCG CAT TCG GTT TTT TTT ACC CTT CTT TAC ACA CTT TTC ATT ATT CTG
MicF binding domain on OmpF:
- AGA CAC ATA AAG ACA CCA AAC TCT CAT CAA TAG TTC CGT AAA TTT TTA TTG ACA GAA CTT ATT GAC GGC AGT GGC AGG TGT CAT AAA AAA AAC CAT GAG GGT AAT AAA TAA TGA TGA AGC GCA ATA TTC TGG CAG TGA TCG TCC CTG C
(Bold is where the primers anneal)
June 16
We used PCR to extract the sRNA gene: MicA and target sequence : ompA. from the e.coli chromosome. We then ran a gel to make sure that we had the right DNA fragments. Our results corresponded to our predictions.
June 17
We are completing a digestion of MicF and OmpF. Following the digestion we will incubate with Shrimp Alkanline Phosphatase and then run a gel to verify the digestion. We will then extract the DNA for the sRNA target sequence and sRNA gene.
