Difference between revisions of "Part:BBa K2387032"
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BBa_K2387032 is created as a means to detect activation of the Cpx pathway of <i>E. coli</i>. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used. | BBa_K2387032 is created as a means to detect activation of the Cpx pathway of <i>E. coli</i>. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used. | ||
| − | + | [https://parts.igem.org/Part:BBa_E0030" eYFP (BBa_E0030)] was cleaved between amino acids 154 and 155 and we fused these N- and C-termini of to the C-terminus of [https://parts.igem.org/Part:BBa_K1486000 CpxR (BBa_K1486000)]. We put these fusions under control of the inducible [https://parts.igem.org/Part:BBa_I0500" pBAD/araC promoter (BBa_BI0500)] to enable controlled protein expression, and strong ribosome binding site (RBS) [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] was placed upstream of the created fusions. This transcriptional unit was constructed and placed in high copy number plasmid [https://parts.igem.org/Part:pSB1C3 pSB1C3] via Golden Gate Assembly. | |
Revision as of 19:47, 30 October 2017
CpxR-eYFPn[1-154] and CpxR-eYPFc[155-238] + araC/pBAD promoter
The [http://parts.igem.com/Part:BBa_K2387005 N-terminus of eYFP] and the[http://parts.igem.com/Part:BBa_K2387006 C-terminus of eYFP] are fused to Cpx response regulator [http://parts.igem.com/Part:BBa_K2387002 CpxR] in order to visualize the activation of the Cpx pathway. Upon activation of the Cpx pathway, CpxR gets phosphorylated by E. coli endogenous CpxA after which it can homodimerize. This protein-protein interaction can be visualized using BiFC, hence the fusion of eYFPn[1-154] and eYFPc[155-238]. The fusion is put under the control of the L-arabinose inducible araC/pBAD promoter. Strong RBS BBa_B0034 is used to regulate transcription.
CpxR-eYFPn and CpxR-eYFPc fusions were linked together using a [http://parts.igem.com/Part:BBa_K1486004 Flexible Linker] consisting of two times the amino acids GGGGS.
This part is used to visualize the activation of the Cpx pathway via Bimolecular Fluorescence Complementation by using the CpxR-CpxR interaction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal XhoI site found at 1283
Illegal XhoI site found at 2498 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1694
Illegal AgeI site found at 2909 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Usage and Biology
BBa_K2387032 is created as a means to detect activation of the Cpx pathway of E. coli. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used.
" eYFP (BBa_E0030) was cleaved between amino acids 154 and 155 and we fused these N- and C-termini of to the C-terminus of CpxR (BBa_K1486000). We put these fusions under control of the inducible " pBAD/araC promoter (BBa_BI0500) to enable controlled protein expression, and strong ribosome binding site (RBS) BBa_B0034 was placed upstream of the created fusions. This transcriptional unit was constructed and placed in high copy number plasmid pSB1C3 via Golden Gate Assembly.