Difference between revisions of "Part:BBa K2282011"

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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average lifetime of 12 seconds stability at 37°C, as opposed to 20 minutes at 15°C, a temperature at which the RNAse is no longer active (Barria et al. 2013).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.

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