Difference between revisions of "Part:BBa K1104102:Design"
 
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　　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.  <br>
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'''FimH Primers:'''
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{|class="wikitable"
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|-
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!
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!primer sequence
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!length(nt)
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!binding part temp.
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!GC%
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|-
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|forward:
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| TTCGCCTGTAAAACCGCCAATG
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|22
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|60℃ ||50%
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|-
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|reverse:
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| AGTAGGCACCACCACATCATTATTG
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|25
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|59℃ ||44%
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|}
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 +
'''Backbone Primers:'''
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{|class="wikitable"
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|-
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!
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!primer sequence
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!length(nt)
 +
!binding part temp.
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!GC%
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|-
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|from RFP reverse:
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| agcaccggtggagtgacgac
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|20
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|63℃ ||65%
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|-
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|from terminator: || taataacgctgatagtgctagtgtagatcgctactag ||37 ||63℃ ||41%
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|}
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　　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.  <br>
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1104102 short</partinfo>
 
<partinfo>BBa_K1104102 short</partinfo>

Latest revision as of 05:45, 30 October 2013

　　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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 781
    Illegal AgeI site found at 893
  • 1000
    COMPATIBLE 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.