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-Inv pnirB-invasin was constructed by extension PCR of the invasin gene from BBa_K299812 (link https://parts.igem.org/Part: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).

Source

The invasin gene from Yersinia is derived from https://parts.igem.org/Part:BBa_K299812 and the pnirB promoter is derived from Jayaraman et al., (1988).

References

The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. Molecular Microbioiogy (1988) 2(4), 527-530