Difference between revisions of "Part:BBa K1804002:Design"
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===Design Notes=== | ===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). | ||
Revision as of 16:38, 19 September 2015
pNirB-Invasin
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal NotI site found at 7 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
- 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16 - 1000COMPATIBLE 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