Team:Wageningen UR/Notebook/Journal/Cpx

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• Cpx Signal Transduction

Cpx Signal Transduction

By: Stijn Prinsen

Here you can find Stijn's work on connecting antigen detection to the Cpx system. Curious about his results? Read more about it here.

May

Week 1 (1st of May - 7th of May)
Got our own office. Started searching literature for a way to design a functioning system.

Week 2 (8th of May - 14th of May)
Commenced the writing of my proposal. Also went to the BCF career event in Utrecht.

Week 3 (15th of May – 21st of May)
I officially started my iGEM thesis and worked full time on my proposal now. Found out being Treasurer is basically a fight against bureaucracy.

Week 4 (22nd of May – 28th of May)
Met with John van der Oost about my project, used his input to finish my proposal at the end of the week. Started figuring out how Snapgene works.

June

Week 5 (29th of May – 4th of June)
Became an intermediate user of Snapgene and applied it to create constructs and design primers. Tried to find usable BioBricks in the iGEM repository.

Week 6 (5th of June – 11th of June)
More Snapgene, redesigned all primers and constructs.

Week 7 (12th of June – 18th of June)
Walked into the lab for the first time and made (slightly) competent cells. Prepared some media and buffers, received a part of my primers and decided to convert the rest of my primers to be Q5-compatible.

Week 8 (19th of June – 25th of June)
Successfully PCR-amplified CpxP gene from the E. coli K12 genome. Also finished designing and ordering all primers for first experiment. I was forced to write a personal facebook post, 'for fun'.

July

Week 9 (25th of June - 2nd of July)
Transformed cells with BioBrick plasmids and isolated plasmids. PCR-amplified the inserts for Golden Gate. Did not manage to amplify the tac promoter from a BioBrick plasmid, sadly.

Week 10 (3rd of July - 9th of July)
Managed to successfully amplify CpxP and Affibody genes in multiple configurations for different fusions. Performed iGEM InterLab study measurements together with Jurre. Finally amplified tac promoter successfully. Attempted construction of pSB1C3: mRFP1 + CpxP using GoldenGate, but failed.

Week 11 (10th of July - 16th of July)
Obtained a plasmid containing MBP from supervisor Prarthana. Amplified different parts of MBP, except the back part, to create MalE31 mutant. Successfully created pSB1C3: mRFP1 + CpxP construct using GoldenGate.

Week 12 (17th of July - 23rd of July)
Multiple CpxP-affibody fusion variants were assembled together with the backbone using GoldenGate. Assembled constructs were transformed to E. coli DH5a.

Week 13 (24th of July - 30th of July)
Amplified Maltose-Binding Protein back part and CpxP-His by PCR. AraC + pBAD promoter was ligated into pSB3T5 backbone and successfully transformed. Made competent cells of E. coli KEIO strain JW5558-1 containing a CpxP knock-out.

August

Week 14 (31st of July - 6th of August)
Amplified whole pSB3T5: AraC + pBAD constructs to have GoldenGate compatible overhangs. Perform GoldenGate reaction with pSB3T5: AraC + pBAD and multiple MBP parts to create pSB3T5: pBAD + MalE31. Later results showed that it was not successful, unfortunately. Also created pSB3T5: pBAD + MBP and pSB1C3: mRFP1 + CpxP-His.

Week 15 (7th of August - 13th of August)
Removed pCpxR 1 bp insertional mutation from pSB1C3: mRFP1 + CpxP-Affibody fusion variants by PCR. Subsequent transformation of fusion variants (without mutations) to JW5558 strain. Repeat of pSB3T5: pBAD + MalE31 construct, again without success.

Week 16 (14th of August - 20th of August)
pSB3T5: pBAD + MalE31 construct creation by GoldenGate was attempted with higher insert ratio, successful.

Week 17 (21st of August - 27th of August)
Double transformations of pSB3T5: pBAD + MalE31 and pSB1C3: mRFP1+CpxP-Aff fusions were performed. The Cpx system induction by KCl was tested in BL21DE3 and CpxP KO strains, but results were confusing. Started cloning the membrane-tethered CpxP-Aff fusions by creating the MalE24-1 gene.

September

Week 18 (28th of August - 3rd of September)
MalE24-1 gene was successfully created. GoldenGate was performed to fuse the MBP misfolder with the CpxP-Aff fusions, resulting in half of the constructs successfully created so far.

Week 19 (4th of September - 10th of September)
Performed two more measurements of Cpx induction with KCl using old protocol, resulting in confusing data again. Tried new protocol: growing induced strains overnight and measuring in morning. This resulted in better data of Cpx induction by KCl.

Week 20 (11th of September - 17th of September)
Repeated transformation of GoldenGate samples to obtain pSB1C3: mRFP1+MalE24-1-CpxP and ”-MalE24-1-CpxP-Aff, Did new measurement protocols with M9 medium, giving better results of Cpx induction by KCl.

Week 21 (18th of September - 24th of September)
Tested the inhibitory capacity of CpxP-Aff fusions using the new protocol in M9 medium. Results showed that variants were able to suppress the Cpx system like native CpxP (except Aff-CpxP-His). Started on Wiki design.

October

Week 22 (25th of September - 1st of October)
Tested whether inducer MalE31 could active Cpx system by dissociating CpxP(-Aff) from CpxA, results indicated that this did not work. Dropped this experiment due to time constraints. Tested the spheroplast protocol, microscopy showed that cells were spheroplasted. More Wiki design.

Week 23 (2nd of October - 8th of October)
Transformed the MalE24-1-CpxP-Aff fusion constructs to E. coli CpxP knock-out strain. Tested the inhibitory capacity of the MalE24-1-CpxP-Aff fusions in spheroplasted cells, results indicated that some of the fusions could suppress the Cpx system. Repeated with replicates to obtain more reliable results. Even more Wiki design.

Week 24 (9th of October - 15th of October)
Did two more measurements on spheroplasted cells, changed the protocol a bit, resulting in better data. Started cloning the ptac+MalE24-1-CpxP-Aff and “-Aff-CpxP constructs to a pSB3T5 backbone for integration of my project with project of Bart. Guess what? Wiki design.

Week 25 (16th of October - 22nd of October)
Continued backbone change for integration with Bart. Did the final measurement on the designed system to see if IgG could induce the Cpx system by binding the membrane tethered CpxP-Aff fusions. Results are inconclusive for reasons unclear. Needs additional verification.

Week 26 (23rd of October - 29th of October)
Biobricks were prepared and shipped. Wiki page for my result was written.