Team:Wageningen UR/Notebook/Journal/BifC

• Project

• Background

• Wet-lab

• Results
• BioBricks

• Model

• Cpx Kinetics
• Quorum Sensing
• Cpx Integration
• QS Integration

• Human Practices

• Integrated HP
• Safety & Ethics
• Collaborations
• Future Perspectives
• Engagement
• Meetup

• Application

• Demonstrate
• Device
• Roll-out Plan

• Notebook

• Protocols
• InterLab
• Lab Journal
• Achievements

• Team

• Members
• Attributions
• Acknowledgements

• 
• Project
• Background
• Wet-lab
• Model
• Human Practices
• Application
• Notebook
• Team

• Wet-lab
• Model
• Application
• Human Practices
• Notebook
• Team
• Parts
• Results
• Cpx Kinetics
• Quorum Sensing
• Cpx integration
• QS integration
• Integrated HP
• Safety & Ethics
• Collaborations
• Future Perspectives
• Engagement
• Meetup
• Demonstrate
• Device
• Rollout Plan
• Protocols
• InterLab
• Journal
• Achievements
• Members
• Attributions

• Home
• Notebook
• Journals
• Cpx Signal Relay

Cpx Signal Relay

By: Bart Scholten

Here you can find Bart's work on the BifC complementation of the Cpx system. Curious about his results? Read more about it here.

May

Week 1 (1st of May - 7th of May)
Getting started, learning how a MacBook works, proposal writing. Doing some course in the mornings as well.

Week 2 (8th of May - 14th of May)
Moved to iGEM Headquarters in the Helix building. Obtained a desk with window view and a PC with a shitty but understandable Windows OS. More proposal writing, pimping my desk and doing some course in the mornings as well.

Week 3 (15th of May – 21st of May)
Finished up proposal, tried to understand how Snapgene and Golden Gate Assembly works and printed the Swanson Pyramid of Greatness to inspire the team. Also went to NBC to show off our project.
Week 4 (22nd of May – 28th of May)
Finally acquired sufficient knowledge on Golden Gate Assembly, designed a load of primers and subsequent constructs and showed of my labskills by making both competent cells and several media.

June

Week 5 (29th of May – 4th of June)
Redesigned my primers and constructs and found out how to validate my experiments. The codon usage table suddenly becomes very fascinating when you need to find silent mutations.

Week 6 (5th of June – 11th of June)
Finalized constructs. Just kidding, improved them even more using Martas feedback, so looked at SnapGene all week.

Week 7 (12th of June – 18th of June)
Ordered all 32 primers, isolated genomic DNA from E. coli K12 to obtain CpxR and CpxA genes and played around with delivered dry ice.

Week 8 (19th of June – 25th of June)
Transformed DH5a with relevant BioBricks (eYFP, TEV protease, araCpBAD promoter) and made PCR products of all CpxA, CpxR, eYFP and TEV protease fragments for experiments 1 and 2. BBa_B10500 araCpromoter didnt work, so opted for BBa_B1321333, based on Martas alignments.

July

Week 9 (26th of June – 2nd of July)
Finished all PCR products for experiment 3 except backbones, sequenced BBa_B10500 and BBa_B1321333 and cleaned up PCR products.

Week 10 (3rd of July – 9th of July)
Ordered new primers for backbones and some good news: pSB1C3-araC-pBAD finally works! Despite this success, pSB3T5 is still fighting back. Spent a whole friday making friends with other European teams in Delft's European iGEM meetup.

Week 11 (10th of July - 16th of July)
Finally obtained pSB3T5 backbone and attempted first GG assemblies to test fluorescence of eYFP and respective termini. Improved yield of pSB1C3-araC-pBAD as well.

Week 12 (17th of July - 23rd of July)
Assembly of pSB3T5-CpxA constructs (no promoter). Assembly of pSB1C3-CpxR constructs.

Week 13 (24th of July - 30th of July)
Assembly of YFP-ZIP constructs and prove the binding affinity. Assembly of pSB1C3-CpxR-TEV. Initial fluorescence test under different levels of induction: eYFP shows fluorescence, the termini do not; hints at a lack of affinity of N and C termini to reassemble. Tried to insert araC-pBAD promoter in CpxA constructs via BioBricks, which failed.

August

Week 14 (31st of July - 6th of August)
Biobrick Assembly still does not work.. Sequencing shows weird inserts. Golden Gate Assembly of pSB1C3-araC-pBAD-eYFPc-cZIP in two steps gave a lot of transformants. Experimented with CpxR-eYFP BiFC based on 2014 eFPL and Marta’s growth protocols; unsuccessfull so far…

Week 15 (7th of August - 13th of August)
Finally got 1C3-araC-pBAD-eYFPc-cZIP and repeated growth experiment (10x dilution) with some moderate success. Optimal KCl induction resembles 2014 EFPL, but no real growth is seen (yet). Went to Munich to see what's up.

Week 16 (14th of August - 21st of August)
Back from Munich, saw what was up. Returned to Winterswijk, beautiful as ever. Back in the Lab, cotransformed CpxAeYFPn and CpxReYFPc for Exp1 and repeated CpxR dimerization, but someone stopped the experiment, which is not really nice. Saw that Leucine Zippers slightly increase affinity of eYFP termini.

Week 17 (22nd of August - 28th of August)
BioBrick might be solved by increasing ligation times extensively, but designed primers to circumvent this anyway. Repeated 2014 EFPL teams result. Got confirmation we can attend the Jamboree in Boston after the Go/No-go presentation.

September

Week 18 (29th of August - 3rd of September)
Transferred constructs to BL21 to improve protein expression (CpxA/CpxR). PCR'ed templates for CpxA-eYFP-ZIP constructs and 3T5 backbone, which were subsequently transformed to DH5a. Repeated CpxR BiFC, but to no avail.

Week 19 (4th of September - 10th of September)
Semi-successful BiFC experiment (induced overnight for a change)! Transformed K12 and K12dCpxR with BiFC CpxR to see if this improves the BiFC result. Searched the whole building for hours for two simple inoculations! Apparently the ASKA collection and KEIO collection do not share the same selection marker. Made two batches of competent cells the old-fashioned way, like an idiot. They were super competent though, as all my plates were overgrown.

Week 20 (11th of September - 17th of September)
Performing BiFC in M9 doesn’t change anything, sadly. Cotransformed TEV and ZIP constructs to BL21 and transformed RnRc cultures to K12 and K12dCpxR. Overflow in BiFC expertiment at the start (how is that even possible)...

Week 21 (18th of September - 24th of September)
Tried BiFC in KCl-less PBS, which didn't work out great (makes sense as it contains a shitload of NaCl). Played around with TEV and ZIP constructs, but José’s lysis protocol didn’t do any miracles unfortunately. He saw similar issues and cloned both YFP-ZIP halves into one cell (which worked for him). Started to suspect that resuspending in PBS might set off BiFC under any condition: measured in uncentrifuged LB cultures. And would you believe it, it worked! Started my 2nd Labjournal (I'm such a hard worker), BiFC CpxR proved to be reproducible!

Week 22 (25th of September - 31st of September)
Let’s give CpxA-CpxR interaction a shot in K12, so transformed accordingly. Had some troubles amplifying necessary products for YFP-ZIP tests. Designed primers to show BiFC modularity by using Venus, which showed the fastest maturation according to José’s experiment. Tested different L-Ara inductions for Sabines models as well. To combine my work with Stijns project, placed RnRc in the 1T3 backbone.

October

Week 23 (1st of October - 7th of October)
Realized placing RnRc in 1T3 is useless, but succesfully cotransformed K12 CpxAeYFPn CpxReYFPc! Dropping a plate with your samples to the ground before measuring significantly makes your results worse, proven by empirical research today. GoldenGate assembly of CpxR-Venus is troublesome, unfortunately.

Week 23 (8th of October - 14th of October)
Successfully proved that CpxA-eYFPn CpxR-eYFPc is not a good interaction to visualize Cpx activation, as expected. Growing E. coli in LB-serum experiments were unsuccessful. We should try a different protocol next time. As leucine zippers do not reassemble in vitro, cloned CpxA-eYFPnnZIP and CpxA-cZIPeYFPc in one plasmid. This turns out to be difficult.