Part:BBa_K1104102:Design
This part is constructed through blunt-end ligation. We have designed two sets of primers. One for the FimH mannose-binding domain from E. coli, and another for the backbone, which has LacI regulated promoter and RFP coding sequence on pSB1A2 plasmid. The primer sets are shown below.
FimH Primers:
| primer sequence | length(nt) | binding part temp. | GC% | |
|---|---|---|---|---|
| forward: | TTCGCCTGTAAAACCGCCAATG | 22 | 60℃ | 50% |
| reverse: | AGTAGGCACCACCACATCATTATTG | 25 | 59℃ | 44% |
Backbone Primers:
| primer sequence | length(nt) | binding part temp. | GC% | |
|---|---|---|---|---|
| from RFP reverse: | agcaccggtggagtgacgac | 20 | 63℃ | 65% |
| from terminator: | taataacgctgatagtgctagtgtagatcgctactag | 37 | 63℃ | 41% |
The design of the primer exactly let the FimH mannose-binding domain coding sequence insert before the stop codon in the RFP coding sequence. This way we have constructed a fusion protein, which the RFP can serve as the signal whether the FimH has bind to the mannose polymer or not.
pLac+RFP-FimH
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
1. During blunt-end ligation, we should add a kinase, in order to phosphoylate the two ends of the DNA that we cloned. The ligase can only work when the DNA is phosphorylated.
2. After ligation, it is a must to do a PCR check to see if you have ligated the coding sequence in the right direction.
Source
This part is ligated from different sources:
1. Escherichia coli K-12 substrain MG1655 genomic sequence
2. Biobrick sent from the headquarter
References
1. EcoCyc: Encyclopedia of Escherichia coli K-12 Genes and Metabolism [http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG10315-MONOMER#/ FimH]
2. Wellens, A., Lahmann, M., Touaibia, M., Vaucher, J., Oscarson, S., Roy, R., Remaut, H., ... Bouckaert, J. (January 01, 2012). The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH. Biochemistry, 51, 24, 4790-9.