Part:BBa_K1804002:Design

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-Invasin

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 2.9 kb product, while lane 4 is the 1 kb ladder.

Figure 2: PCR of the 2.9 kb 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 FP_BBP_Junk and RP_BBS_Junk primers, 1kb ladder.

Figure 3: Colony PCR with BioBricks Prefix and Suffix 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.