Part:BBa_K098997
cIts coding region
This it the temperature sensitive cI from the PGW7 plasmid. It has the Elowitz RBS in front (BBa_B0034). It produces a temperature sensitive cI repressor that loses binding efficiency with the cI promoter between 35 and 42 degrees C.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization IONIS_PARIS 2017
Group & Author: IONIS_PARIS /La Paillasse/2017
Summary:
We have use the part BBa_K098997 in our part BBa_K2282013 to build the heat shock response. We introduce the mRFP to check if the system works at 37ºC and not under 20ºC (used as a reporter). We sequenced our sequence 9 that appears to be red at 37ºC and we saw that the gene cIts was in the sequence. Our part BBa_K2282013, can be used as a gene reporter to show visually if the clts gene is present in the bacteria, thanks to the mRFP. We send the part BBa_K2282013 for sequencing at GATC and this is the result:
Method:
See the bacterial transformation protocol in our wiki IONIS_PARIS 2017_Lab work_protocols.
We decided to sequence the biobrick BBa_K2282013 by GATC in order to testified the presence of the whole integrity of the sequence (cI lambda reporter gene). The results were successful, the part BBa_K2282013 was well sequenced.
BBa_K2282013:
Forward Primer:
CNNGCGCG TTGGCCG ATTCATT AATGCA GCTGGCAC GACAGGTTTCCCG ACTGGAAAGCGGGC AGTGAGCGCAACGCAA TTAATGTGA GTTAGCTCACTCAT TAGGCACCCCAGG CTTTACACTTTATGC TTCCGGCTCGTAT GTTGTGTGGAATTG TGAGCGGATAACAA TTTCACACATACTA GAGAAAGAGGAGAA ATACTAGATGGC TTCCTCCGAAGAC GTTATCAAAGAGTT CATGCGTTTCAAA GTTCGT ATGGAAGGTTC CGTTAACGGTC ACGAGTTCGAAA TCGAAGGTGA AGGTGAAGGTCGTC CGTACGAAGGTA CCCAGACCGCTA AACTGAAAGTT ACCAAAGGTGGT CCGCTGCCGTTC GCTTGGGACATCC TGTCCCCGCAGTT CCAGTACGGTTCCA AAGCTTACGTTAAA CACCCGGCTGAC ATCCCGGACTACC TGAAACTGTCCTT CCCGGAAGGTTTC AAATGGGAACGTG TTATGAACTTCGAA GACGGTGGTGTTG TTACCGTTACCCAGG ACTCCTCCCTG CAAGACGGTGAG TTCATCTACAAAGTT AAACTGCG TGGTACCAACT TCCCGTCCGAC GGTCCGGTTA TGCAGAAAAA AACCATGGGTTG GGAAGCTT CCACCGAACGTAT GTACCCGGA AGACGGTGC TCTGAAA GGTGAAATCAA AATGCGTCT GAANCTGAA AGACGGTGGTCA CTACGACGCTGA AGTTAAAACCA CCTACATGGCTA AAAANCCG GTTCAGC TGCCG GGTGCTTACA AAACCGACATCAAA CTGGACATCACCTC CCACAACGAAGACTA CACCATCGTTG AACAGTACGAAC GTGCTGAAGGTCGT CACTCCACCGGTGC TTAATAACGCTGATA GTGCTAGTGTAGATC GCTACTAGAGCCAGG CATCAANTAAAACGAA AGGCTCAGTCGAA AGACTGGGCCTT TCGTTTTAT CTGTTGTTTGTCG GTGAACGCTC TCTACTANAGT CNNNCGGGCTC ACCTTCGG NGGGNCCTT TCTGCG
Figure 1: Sequencing by GATC part BBa_K2282013 (forward primer).
Reverse Primer:
CCNG AGGTGAGCC AGTGTGACT CTAGTAGAGAG CGTTCACC GACAAACAA CAGATAAAACG AAAGGCCCAGT CTTTCGACT GAGCCTTTCG TTTTATTTGA TGCCTGGCTC TAGTAGCGATC TACACTAGCA CTATCAGCG TTATTAAGCA CCGGTGGAGT GACGACCTTCA GCACGTTCGT ACTG TTCAA CGATG GTGTAGTCTT CGTTGTGGGAGG TGATGTCCAGTTT GATGTCGGTTTTG TAAGCACCCGGCA GCTGAACCGGTTT TTTAGCCAT GTAGGTGGTTT TAACTTCAGCGTC GTAGTGACCACC GTCTTTCAGTTTC AGACGCAT TTTGATTTCAC CTTTCAGAGCA CCGTCTT CCGGGTACAT ACGTTCGGT GGAAGCTTCCC AACCCATGGT TTTTTTCTGCAT AACCGGACCGT CGGACGGGAAGTT GGTACCACGCAG TTTAACTTTGT AGATGAACTC ACCGTCTTG CAGGGAGGAG TCCTGGGTA ACGGTAACAA CACCACCGT CTTCGAAGT TCATAACACGT TCCCATTTGA AACCTTCC GGGAAGGACA GTTTCAGGT AGTCCGGG ATGTCAGC CGGGTGTT TAACGTAAG CTTTGGAACCG TACTGGAACTG CGGGGACAG GATGTCCC AAGCGAA CGGCAGCGG ACCACCTTTG GTAACTTTC AGTTTAGCG GTCTGGG TACCTTCG TACGGACGAC CTTCACCTTC ACCTTCGAT TTCGAACTC GTGACC GTTAACGGAA CCTTCCATAC GAACTTTGAA ACGCATGAACT CTTTGATA ACGTC TTCGGAGGA AGCCAT CTAGTATTTCT CCTCTTTCTCT AGTATGTGTGAA NTTGTTATC CGCTCACAATT CCACACAACATA CGAGCCGG AAGCATAANGT GTAAANCCNG GGGGTGCCTA ATGGAGNGAG CTAACNTCACATT NAATTGGCGTTG NNGCTCACT GGCCCGCT TTTCCAGT CCGGAA
Figure 2: Sequencing by GATC part BBa_K2282013 (reversed primer).
Results:
The bacterial transformation worked and the colonies presenting the gene clts were red thanks to the mRFP. So in conclusion, we could use this part as a gene reporter, to show that clts is present pSB1C3.
Characterisation of BBa_K2282013 (pL-mRFP regulated by cI857 repressor) was done with the control BBa_K2282012 (pL-mRFP constitutive). The BBa_K2282012 mRFP is supposed to be expressed at any temperatures whereas the BBa_K2282013 mRFP expression is meant to be regulated by the cI857 repressor and expressed only above 30°C.
We did a kinetic of mRFP expression for both BBa_K2282013 and BBa_K2282012 at 18°C and 37°C to know if our system worked. We needed these data because of the negative visual results we had previously in our lab. The manipulation was carried out thanks to our advisor Nicolas Cornille, who used a Tecan microplate reader. We could not use the fluorescence measurement because the device did not have the module to do so. The absorbance measurement was hence did at 500nm for the mRFP and the bacterial concentration was assessed with OD800. The device did measurements every 12 minutes during 60 hours. Here are the results.
IMPROVEMENT REFERENCE: IONIS_PARIS 2017
Our part BBa_K2282013 contains the valuable part BBa_K098997 corresponding to the cI lambda repressor. Even though, the mRFP inserted inside our BBa_K2282013 part could be used as a reporter gene to show visually the successful transformation of the cI lambda repressor gene in E.coli. That way, when an iGEM team wishes to characterize the part BBa_K098997 they can use our part and will not need to do PCR colony to select the right transformed colony. However, the part BBa_K2282013 does not report the expression of the cI lambda repressor gene, but only its presence.
The part used to improved BBa_K098997 (Group: iGEM08_Harvard) is: BBa_K2282013 (Group: iGEM17_IONIS-PARIS), Designed by:Eliott LAFON.
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