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average lifetime of 12 seconds stability at 37°C, as opposed to 20 minutes at 15°C, a temperature at which the RNAse is no longer active (Barria et al. 2013).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 : https://parts.igem.org/Part:BBa_K328001

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.

Characterization

Summary: The cold-shock plasmid was obtained by ligating the BBa_K2282011 part into the pSB1A3 backbone and was characterize through visual observations and kinetics absorbance assays. For more details go to our wiki IONIS_PARIS 2017 (lien characterization).

Methods:

We are using the blue chromoprotein amilCP with the DS box (part BBa_K2282006) as a reporter gene.

1) Visual observation assays:

Transformed bacteria were pre-incubated at 37°C until the O.D.600nm reached 0.5. They were then cultured at different temperatures (12°C, 15°C, 20°C, 27°C) with a control at 37°C for different incubation times (18h, 20h, 42h) (LINK protocol).

amilCP expression at 15°C:

Figure 1: Pellets of bacteria transformed with BBa_K2282011, after incubation at 37°C for 18h (on the left) and 15°C for 18h (on the right).

Figure 2: Pellets of bacteria transformed with BBa_K2282011, after incubation at 37°C for 42h (on the left) and 15°C for 42h (on the right).

amilCP expression at 20°C:

At 20°C after 20h, bacteria transformed with BBa K2282011 were also blue compared to the wild type bacteria. This result shows the cold response system doesn’t react at a precise temperature. It’s efficiency likely increases as much as temperature decreases.

Figure 3: Pellets of bacteria transformed with BBa_K2282011, after incubation at 20°C for 20h (on the left) and 37°C for 20h (on the right).

amilCP expression at 27°C:

In order to get a better insight into the cold-shock expression pattern, the experiment was also carried out at the intermediary temperature of 27°C. Bacteria were slightly blue for 27°C incubation time and almost uncolored at 37°C. Despite the presence of a light coloration, this results shows the significant expression reduction at 27°C compared to 20°C.

Figure 4: Pellets of bacteria transformed with BBa_K2282011, after incubation 20h at 27°C (on the left) and 20h at 37°C (on the right).

Figure 5: Pellets of DH5α transformed with BBaK2282011, after incubation 20h at respectively 12°C, 15°C, 20°C, 27°C and 37°C (on the left), pellets of DH5α transformed with BBaK2282005 after incubation 20h at respectively 15°C and 37°C (middle), and pellets of wild type DH5α (on the right).

Visual observations confirmed that our part BBa_K2282011 works as expected and allows efficient protein production only at temperatures starting from about 20°C, until low temperature as 12°C. Moreover the cold response system blocks greatly but not completely the protein expression above 27°C.

Considering the material available in our lab, amilCP was a useful reporter gene for observing the results with the naked eye. But it was complicated to perform OD measurements without extracting proteins because amilCP has its maximum of absorbance at 588nm near to 600nm, the wavelength used to measure the bacteria concentration. This proximity is prone to alter the actual signal corresponding to the amilCP protein.

2) Kinetics measurements assays:

E.coli BL21 were transformed with either BBa_K2282011 coding for the cold-dependent amilCP expression, or BBa_K2282005 coding for constitutive amilCP expression. They were cultivated at 37°C for 60 hours and OD588 was measured every 10min approximately with a Spark 10M Tecan.

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

We decided to sequence the biobrick BBa_K2282011 by GATC. 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

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:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]