Part:BBa_K2282013

mRFP under heat inducible system pL/cI857

This part codes for the full sequence of mRFP, a red fluorescent protein, above 37°C. The construct is composed of a cI857 coding gene behind the strong constitutive promoter J23100 and RBS and an mRFP behind a pL promoter that is regulated by the gene cI857 and RBS. Both cI857 and mRFP coding genes are in front of a terminator. Leftward promoter (pL) derived from phage ƛ is fully repressed at low temperature by the thermolabile cI857 protein. cI857 loses repression activity on the pL promoter when the temperature rise above 30°C until 42°C, when it can no longer bind to pL. The aim of this construct is to code for mRFP when the temperature rises above 30°C, while the transcription is repressed under 30°C.

Source of this part

BBa_K2282013 part has been assembled to the pL promoter - RBS - Double terminator (E.coli his operon terminator). This double terminator was extracted from the strain E.coli K12 MG1655 complete genome, size length 4,641,652 pb in order to be different from the one in the part BBa_K22820012 and avoid concatemers problems.

Design consideration

This part sums up our final heat sensitive construction. The overall strategy we chose is complex and we were not sure it would work. Our bibliographic review and analysis of the Paris-Saclay iGEM 2017 project (using DNA thermometers) we decided to use strong promoters (Anderson BBa_J23100 with its respective RBS and pL promoter) to obtain higher expression levels. We had a slight problem in sequence design with IDT because their system detected our double use of double terminators as concatemers and did not let us introduce the same double terminator BBa_B0015 so we change the double terminator of the new part by an E.coli terminator operon.

References

Norma A Valdez-Cruz, Luis Caspeta, Néstor O Pérez, Octavio T Ramírez, Mauricio A Trujillo-Roldán - Production of recombinant proteins in E.Coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters - Microbial Cell Factories 2010, 9:18.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1745
Illegal AgeI site found at 1857
1000
COMPATIBLE WITH RFC[1000]

Characterization IONIS_PARIS 2017

Group & Author: IONIS_PARIS /La Paillasse/2017

Summary: We wanted to study if our part BBa_K2282013 worked as predicted. This was the second step of our proof of concept : expressing a specific compound (here the chromoprotein mRFP) at high temperatures only (above 30°C) (Winstanley et al 1989,Valdez-Cruz et al 2010).

We decided to sequence the Biobrick BBa_K2282013 by GATC in order to testify the presence of the whole integrity of the sequence. The results were successful, the part BBa_K2282013 was well sequenced. See the results on the registry here (https://parts.igem.org/Part:BBa_K2282013).

Characterization of BBa_K2282013 (pL-mRFP regulated by cI857 repressor) was done with the control BBa_K2282012 (pL-mRFP constitutive).

Method: We first characterize our part by doing a bacteria culture (BBa_K2282013 - pSB1C3) at 37ºC. We took the DO until it reached 0,5. After that we split it into two falcons, one was stocked at 15ºC and the second one was stoked at 37ºC. After 20h00 of incubation the results were not successful, both colonies were red at 15ºC and 37ºC.

We supposed that when the system heat is activated (red color due to the mRFP), it can not represses the action of the mRFP even if the system is in cold shock temperatures. The problem is that we can not see very well if our part works on high temperatures or not. For the characterization we have always used the part BBa_K2282012 as a control.

We try do change the characterization by making our bacterial transformation with the part BBa_K2282013 growth at room temperature (around 20ºC), normally we should have white colonies in other to start again the characterization and do not perturb the first results.

Figure 1: BBa_K2282013 grown at 25ºC (room temperature).

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.

Parts BBa_K2282012 / BBa_K2282013 /WT at 37°C

First of all, the blue line corresponds to the ratio Abs500/OD800 from the wild-type DH5α. It is meant to represent the background noise that the Tecan measured. What we can see here is that both BBa_K2282012 and BBa_K2282013 mRFPs are expressed at 37°C, which is consistent. At 37°C the cI857 repressor has been reported to be denaturated (switch from dimer to monomer) and no longer binds to the pL promoter, permiting the mRFP expression.

Parts BBa_K2282012 / BBa_K2282013 /WT at 18°C :

Here several information are to be interpreted. First both BBa_K2282012 and BBa_K2282013 are expressed at 18°C. This is not consistent with what we said previously and confirms the negative visual results we had previously. This shows hence that BBa_K2282013 is not working properly. This could be due to the biobrick construction itself. Indeed, in BBa_K2282013, the reading frame of the cI857 is in the 5’ 3’ orientation, whereas the mRFP reading frame is in the 3’5’ orientation. This had to be done because IDT could not support the double biobrick construction. Both cI857 and mRFP from BBa_K2282013 had therefore to share the same terminator.

We can see in any case that BBa_K2282012 works fine at both 37°C and 18°C. Another information is that both mRFP from BBa_K2282012 and BBa_K2282013 are less expressed at 18°C than at 37°C, which is consistent judging that E.coli growth and protein expression is not optimal at 18°C. The considerable standard deviation and the apparent lowering of the DH5ɑ WT curve is due to little evaporation of the culture medium.

We decided to sequence the biobrick BBa_K2282013 by GATC in order to testified the presence of the whole integrity of the sequence. The results were successful, the part BBa_K2282013 was well sequenced.

BBa_K2282013: Forward:

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 2: Sequencing by GATC part BBa_K2282013 (forward primer).

Reverse:

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 3: Sequencing by GATC part BBa_K2282013 (reversed primer).

IMPROVEMENT REFERENCE: IONIS_PARIS 2017

However, as we saw that our biobrick doesn't work, we thought about a possible reconversion for this part. Indeed, this part contains the valuable part BBa_K098997 corresponding to the cI lambda repressor. Even though, the part BBa_K2282013 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 (https://parts.igem.org/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 improved is: BBa_K098997 (Group: iGEM08_Harvard), Designed by: Harvard iGEM 08.

REFERENCES

- Winstanley et al., “Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system”, Appl Environ Microbiol. 1989 Apr; 55(4): 771–777.

- Valdez-Cruz et al., “Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters”, Microb Cell Fact. 2010; 9: 18.