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Team:Illinois/SgrS
From 2009.igem.org
SgrS Target-GFP Fusion
People in Group: Francis Lee, Harsh Shah, Dave Korenchan
Purpose: We want to fuse the SgrS target sequence from the PtsG 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 SgrS gene. We expect to see translational repression of GFP by the small RNA SgrS.
Protocol(s) Used: (links to protocols page)
Recipe(s) Used:
sgrS Gene Sequence: (K-12 substr. MG1655: 77366-77623)
GATGA AGCAA GGGGG TGCCC CATGC GTCAG TTTTA TCAGC ACTAT TTTAC CGCGA CAGCG AAGTT GTGCT GGTTG CGTTG GTTAA GCGTC CCACA ACGAT TAACC ATGCT TGAAG GACTG ATGCA GTGGG ATGAC CGCAA TTCTG AAAGT TGACT TGCCT GCATC ATGTG TGACT GAGTA TTGGT GTAAA ATCAC CCGCC AGCAG ATTAT ACCTG CTGGT TTTTT TTATT CTCGC CGCGC TAAAA AGGGA ACGTA TG
ptsG 5' UTR Sequence: (K-12 substr. MG1655: 1156990-1157166)
AAATA AAGGG CGCTT AGATG CCCTG TACAC GGCGA GGCTC TCCCC CCTTG CCACG CGTGA GAACG TAAAA AAAGC ACCCA TACTC AGGAG CACTC TCAAT TATGT TTAAG AATGC ATTTG CTAAC CTGCA AAAGG TCGGT AAATC GCTGA TGCTG CCGGT ATCCG TACTG CCTAT C
Primers Used:
- Forward/sense sgrS (sRNA gene) primer: 5'p+GATGAAGCAAGGGGGTGCCC
- Reverse/antisense sgrS (sRNA gene) primer: GTTTTTTCTAGACATACGTTCCCTTTTTAGCG
- Forward/sense ptsG (sRNA target sequence) primer: GTTTTTATGCATAAATAAAGGGCGCTTAGATG
- Reverse/sense ptsG (sRNA target sequence) primer: GTTTTTGCTAGCGATAGGCAGTACGGATAC
PCR Products:
sgrS GENE (XbaI restriction site)-269bp:
GATGA AGCAA GGGGG TGCCC CATGC GTCAG TTTTA TCAGC ACTAT TTTAC CGCGA CAGCG AAGTT GTGCT GGTTG CGTTG GTTAA GCGTC CCACA ACGAT TAACC ATGCT TGAAG GACTG ATGCA GTGGG ATGAC CGCAA TTCTG AAAGT TGACT TGCCT GCATC ATGTG TGACT GAGTA TTGGT GTAAA ATCAC CCGCC AGCAG ATTAT ACCTG CTGGT TTTTT TTATT CTCGC CGCGC TAAAA AGGGA ACGTA TGTCT AGAAA AAAC
ptsG 5' UTR (Mph1103I, NheI restriction sites)-200bp:
GTTTT TATGC ATAAA TAAAG GGCGC TTAGA TGCCC TGTAC ACGGC GAGGC TCTCC CCCCT TGCCA CGCGT GAGAA CGTAA AAAAA GCACC CATAC TCAGG AGCAC TCTCA ATTAT GTTTA AGAAT GCATT TGCTA ACCTG CAAAA GGTCG GTAAA TCGCT GATGC TGCCG GTATC CGTAC TGCCT ATCGC TAGCA AAAAC
June 15
PCRs of sRNA genes and target sequences were performed using E. coli extracted chromosomal DNA and primers.
June 16
We ran a gel on our PCR reactions, and the PCRs were successful! The SgrS gene falls at about the 300 bp region on our gel, which agrees with our data for the length of the SgrS gene segment. The PtsG target sequence is smaller than the SgrS gene on the gel, which also agrees with our data.
