Difference between revisions of "Part:BBa K1804002:Design"

(→‎Design Notes)
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===Source===
 
===Source===
  
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The invasin gene from Yersinia is derived from BBaK299812[https://parts.igem.org/Part:BBa_K299812] and the pnirB promoter is derived from Jayaraman et al. (1988).
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The invasin gene from ''Yersinia'' is derived from BBa_K299812 [https://parts.igem.org/Part:BBa_K299812] and the p''nirB'' promoter is derived from Jayaraman et al. (1988).
  
 
===References===
 
===References===
 
Jayaraman, P. S., Gaston, K. L., Cole, J. A., & Busby, S. J. W. (1988). The ''nirB'' promoter of ''Escherichia coli'': location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. ''Molecular microbiology'', 2(4), 527-530.
 
Jayaraman, P. S., Gaston, K. L., Cole, J. A., & Busby, S. J. W. (1988). The ''nirB'' promoter of ''Escherichia coli'': location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. ''Molecular microbiology'', 2(4), 527-530.

Revision as of 08:38, 20 September 2015

pNirB-Invasin


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NotI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Design and Construction of pnirB-Inv

pnirB-invasin was constructed by extension PCR of the invasin gene from BBa_K299812 using BBPrefix-pnirB as the forward primer and a reverse primer for the end of invasin fused to BBSuffix (invendBBSuffixLonger) (Figure 1). BBPrefix-pnirB-invasin-BBSuffix was then amplified into Junk-BBPrefix-pnirB-invasin-BBSuffix-Junk using FP_BBP_Junk and RP_BBS_Junk (Figure 2). Junk-BBPrefix-pnirB-invasin-BBSuffix-Junk was then cloned into pSB1C3 by restriction digest with EcoRI and PstI followed by ligation and transformation into E. coli BL21. The clone was confirmed with colony PCR of Biobricks Prefix/Suffix primers and internal primers invlloF1/InvendBBsuffixlonger (Figure 3).

Figure 1: PCR of the template plasmid BBa_K299812 was performed with BBPrefixpNirBinv and invendBBSuffix to add the NirB promoter to the invasin fragment. Lanes 1-3 show the 2.9kb product, while lane 4 is the 1kb ladder.


Figure 2: PCR of the 2.9kb product to add short overhangs for Restriction digest to aid cloning. 1kb ladder Lane 1-7 PCR of 2.9kb fragment from Figure 1 with Junk_BBPrefix and BBSuffix_Junk primers 1kb ladder.


Figure 3: Colony PCR with Biobricks Suffix/prefix primers, and internal primers to confirm if the clone contains the pnirb-invasin insert in vector.1kb ladder Lane 1-2 Colony PCR of colony 4 with negative control of water for Biobricks Prefix/Suffix primers Lane 3-4 Colony PCR of colony 4 with negative control of water for invlloF1/InvendBBsuffixlonger.


The sequences of the primers used can be found here: [1]. Please refer to our Protocols page [http://2015.igem.org/Team:SPSingapore/Protocol#p13] for details on a mammalian cell invasion assay. Our experimental procedures can be found at our Notebook page [http://2015.igem.org/Team:SPSingapore/Notebook].

Source

The invasin gene from Yersinia is derived from BBa_K299812 [2] and the pnirB promoter is derived from Jayaraman et al. (1988).

References

Jayaraman, P. S., Gaston, K. L., Cole, J. A., & Busby, S. J. W. (1988). The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. Molecular microbiology, 2(4), 527-530.