Part:BBa_K2282011

AmilCP with DSbox under Upelmt/CspA promoter + 5'UTR

Usage and Biology

The UP element is believed to stimulate the transcription of the CspA gene at a cold temperature, although some reports conclude the opposite (Phadtare et al., 2005). This promoter plays a role in transcriptional regulation, but not in translation regulation (DSBox, 5’UTR). The CspA promoter is considered a strong promoter (Mitta et al., 1997). The DSbox is a cis-acting mRNA element enabling assembly of the translation pre-initiation complex, likely through interaction with ribosomal protein S1. The 5’ UTR contains a regulatory sequence called “Cold Box”​ that plays an important role in the stabilisation of the mRNA at low temperature (ATTAAA) (Mitta et al, 1997). The structure of this 5’UTR is extremely unstable at high temperature, as it is believed to be hydrolysed by an RNAse, leading to an average lifetime of 12 seconds stability at 37°C, as opposed to 20 minutes at 15°C, a temperature at which the RNAse is no longer active (Barria et al. 2013). All these elements are thought to act synergistically to induce cold-only expression of the amilCP protein.

Source of this part

We used all the sources of our previous parts (BBaK2282006 and BBa_K2282004). Sequences of the 5’UTR, DSBox and other cold-response elements are very close to the parts of the iGEM10_Mexico-UNAM-CINVESTAV that can be found here : http://partsregistry.org/Part:BBa_K328003

Design consideration

This part sums up our final cold regulated construction. A lot of planning was done to develop our strategy and sequences. The overall strategy we chose is complex and we were not sure if it would work. Through bibliographic study of open source literature, the iGEM Parts Registry, and industrial patents, we saw that the iGEM10_Mexico-UNAM-CINVESTAV team attempted the same strategy as us, but nothing was said on their wiki regarding their results. We also saw that the pCOLD vector from the company Takara also used the same technology, with some variations, and was patented. We therefore thought that this strategy had a good chance of success.

Below is presented the 3D structure of the cspA 5'UTR as computed by SimRNA (a coarse grained statistical potential method for RNA folding simulations). Highlighted in yellow is the DS box sequence, which is important for increased expression levels at low temperatures, and forms part of the coding sequence.

Characterisation

We decided to sequence the biobrick BBa_K2282011 by GATC in order to testified the presence of the whole integrity of the sequence (amilCP DS Box). The results were successful, the part BBa_K2282011 was well sequenced.

BBa_K2282011:

Forward Primer:

GCGTGGCTTAATACGGTTTGACGTACAGAC CATTAAAGCAGTGTAGTAAGGCAAGTCCC TTCAAGAGTTATCGTTGATACCCCTCGT AGTGCACATTCCTTTAACGCTTCAAA ATCTGTAAAGCACGCCATATCGCCG AAAGGCACACTTAATTATTAAAGGT AATACACTATGAGTATGACTGG TATCGTAGTGATCGCTAAACAAAT GACCTACAAGGTTTATAT GTCAGGCACGGTCAATGG ACACTACTTTGAGGTCGAAGGC GATGGAAAAGG TAAGCCCTACGAGGGGGA GCAGACGGTAAAGCTCACTGT CACCAAGGGCGGACCTCTGC CATTTGCTTGGGATATTTTA TCACCACAGTGTCAGTACG GAAGCATACCATTCACCAAG TACCCTGAAGACATCCCTG ACTATGTAAAGCAGTCAT TCCCGGAGGGCTATACAT GGGAGAGGATCATGAA CTTTGAAGATGGTG CAGTGTGTACTGTCAGC AATGATTCCAGCATCCAAG GCAACTGTTTCATCTAC CATGTCAAGTTCTCTGGT TTGAACTTTCCTCCCA ATGGACCTGTCATGCA GAAGAAGACACAGGG CTGGGAACCCAACAC TGAGCGTCTCTTTGCA CGAGATGGAATGCTGC TAGGAAACAACTTTATG GCTCTGAAGTTAGAA NGAGGCGGTCACTA TTTGTGTGAATTTNA AACTACTTACAAGGCAAA GAAGCCTGTGAAGATGCC AGGGTATCACTATGTTGACC GCAAACTGNATGTAACCAAT CACAACAAGGATTACACTTCG GTTGAGCAGTGTGAAATTT CCATTGCACGCAAACCTGN GGTCGCCTAATAACCAG GCATCAAATAAANCGA

Figure 1: Sequencing part BBa_K2282011 with Forward primer

Reverse Primer:

TGAGCCNGTGTGA CTCTAGTAGAGAGCG TTCACCGACAAACAA CAGATAAAACGAAAG GCCCAGTCTTTCGA CTGAGCCTTTCGTTTT ATTTGATGCCTGGTTAT TAGGCGACCACAGGTT TGCGTGCAATGGAAATT TCACACTGCTCAACCGA AGTGTAATCCTTGTTGTG ATTGGTTACATCCAGTT TGCGGTCAACATAG TGATACCCTGGCATCTT CACAGGCTTCTTTGCCTTG TAAGTAGTTTTAAATTCA CACAAATAGTGACCG CCTCCTTCTAACTTCAG AGCCATAAAGTTGTTTCCT AGCAGCATTCCATCTCGTGCA AAGAGACGCTCAGTGTTGG GTTCCCAGCCCTGTGTCT TCTTCTGCATGACAGGTC CATTGGGAGGAAAGTTCA AACCAGAGAACTTGACATG GTAGATGAAACAGTTGCCT TGGATGCTGGAATCATTG CTGACAGTACAC ACTGCACCATCTTCAAAGT TCATGATCCTCTCCCATGTA TAGCCCTCCGGGAATGAC TGCTTTACATAGTCAGG GATGTCTTCAGGGTACT TGGTGAATGGTATGCTT CCGTACTGACNCTGNG GTGATAAAATATCCCAA GCAAATGGCAGAGG TCCGCCCTTGGNGA CAGTGAGCTTTACCG TCTGCTCCCCNNCNT ANGGCTTACCTTTTTCCAT CGCCTTCGACCTCAAAG TAGTGTCCATTGACCG TGCCTGACATATAA ACCTNGNANGNCATT TGNTTANCGATCACTA CNATACCAGTCATA CTCNTAGTGTATTACCT TNNTAATTAAGTG TGCCTTTCGGCG ANNNNNCGTGCT TTACAGATTTTGA AGCGTTAAAG

Figure 2: Sequencing part BBa_K2282011 with Reverse primer

Pre-incubation of transformed bacteria were set up at 37°C until the O.D. reached 0.5. Then they were cultured at different temperatures (12°C, 15°C, 20°C) with a control at 37°C

After centrifuging bacteria, the pellet of bacteria transformed with BBa K2282011 appeared blue at 15°C but not at 37°C. These results were in accordance with the expected results. We tried 18h incubation and 42h to see any change in intensity but it seemed that there was no difference. Therefore, further experiments were only carried out during 20h.

The experiment was also carried out at 18°C and 12°C in an attempt to determine a shift.

At 20°C after 20°C, bacteria transformed with BBa K2282011 were also blue compared to the wild type bacteria.

Same results at 12°C.

The shift should therefore occur between 20°C and 37°C. Plus, the regulatory machine is active until at least 12°C.

Figure 3: OD/Luminescence over the time (h) (BL21 abs588/OD800)

This graph has been made using the same protocol at only 37°C and O.D. 588 measurements were done every 10mn. We compared AmilCP expression between wild type E. Coli, transformed with cold shock plasmid and transformed with constitutive AmilCP inducing plasmid. The results show that the cold shock plasmid (BL21 seq7 abs588/OD800) induces lower expression at 37°C compared to the constitutive one (BL21 seq1 abs588/OD800). It is in accordance with the CspA construction and the previous picture.

Note: O.D.800 has been used as 588 was too much close to O.D.600. For more details you can check our laboratory work in the wiki http://2017.igem.org/Team:IONIS-PARIS

IMPROVEMENT REFERENCE: IONIS_PARIS 2017

Our part BBa_K2282011 contains the valuable part BBa_K328001 corresponding to the amilCP. Even though, the part BBa_K2282011 has been study to demonstrate that introducing the DS box inside the amilCP does not change the molecular structure and function of the amilCP (no negative repercussions). However, the part BBa_K2282011 we don't know yet with accuracy if the DS box could alter the amilCP expression.

The part we improve is: BBa_K592009 (Group:iGEM11_Uppsala-Sweden), Designed by:Lei Sun.

References

Phadtare S, Severinov K. Extended −10 Motif Is Critical for Activity of the cspA Promoter but Does Not Contribute to Low-Temperature Transcription. Journal of Bacteriology. 2005;187(18):6584-6589. doi:10.1128/JB.187.18.6584-6589.2005.

Masanori Mitta et al, Deletion analysis of cspA of Escherichia coli requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction, Molecular Microbiology (1997) 26(2), 321–335

C. Barria et al, “Bacterial adaptation to cold”, Microbiology (2013), 159, 2437–2443

Sequence and Features

Assembly Compatibility:

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• 12
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  Unknown
• 25
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  Unknown
• 1000
  COMPATIBLE WITH RFC[1000]