Difference between revisions of "Part:BBa K2387032"
(→Usage and Biology) |
(→Results - L-arabinose inducibility) |
||
| Line 34: | Line 34: | ||
We perform all experiments in <i>E. coli</i> K12. We grow the cells in saltless LB and induce protein expression with a range of 0.02 - 0.2% L-arabinose. CpxR dimerization and subsequent fluorescence is measured over time, and the system is activated at t=20 min with 75 mM KCl. Check out the full protocol [https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf here]. | We perform all experiments in <i>E. coli</i> K12. We grow the cells in saltless LB and induce protein expression with a range of 0.02 - 0.2% L-arabinose. CpxR dimerization and subsequent fluorescence is measured over time, and the system is activated at t=20 min with 75 mM KCl. Check out the full protocol [https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf here]. | ||
| − | + | <HTML> | |
| + | |||
| + | <div class="figure-center"> | ||
| + | <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/e/e4/T--Wageningen_UR--CpxR_dimerization_araC.png" /> | ||
| + | <div class="figure-center-caption"> | ||
| + | <b>Figure 3:</b> CpxR dimerization visualized with different L-arabinose concentrations over time. | ||
| + | </div> | ||
| + | </div> | ||
| + | </HTML> | ||
Revision as of 20:13, 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.
Results - L-arabinose inducibility
We perform all experiments in E. coli K12. We grow the cells in saltless LB and induce protein expression with a range of 0.02 - 0.2% L-arabinose. CpxR dimerization and subsequent fluorescence is measured over time, and the system is activated at t=20 min with 75 mM KCl. Check out the full protocol here.
