Difference between revisions of "Team:KU Leuven/Notebook/Newsfeed"

-Because two colony of the devices containing the promoter J23117 did not give the expected bands after restriction mapping, we repeated our fluorescence and absorbance measurements with other colonies. In the end, all the selected colonies for the data analysis were confirmed by restriction mapping.
-Last week, we constructed the biobricks LuxI, LuxR and IlvE by performing a phusion high-fidelity tail-PCR on our gBlocks. This week, we digested our biobricks LuxI, LuxR and IlvE with BcuI and EcoRI and purified this with help of a GeneJET PCR Purification Kit of Thermo Fisher Scientific. We ligated this with cutted pSB1C3 by use of T4 ligase. The ligated plasmid was electroporated in E. cloni. After performing a colony PCR and restriction mapping, we concluded that the biobricks LuxR and IlvE were correct. So, we still need to repeat the construction of the LuxI biobrick.
-We transformed Ag43 originating from the iGEM 2015 kit in E. cloni. Thereafter, we made a liquid culture and mini prepped this.
-This Friday the Gibson Assembly was repeated, but this time a pUC19 vector was added. This vector was first modified by tail PCR in this way that the overhangs would match those of the gBlocks. This DNA was electroporated in E. cloni.

-This week we made electrocompetent cells. Their transformation efficiency is higher than that of chemocompetent cells, so they could be useful in making biobricks and transforming our assembled gBlocks.
-We repeated the tail PCR of luxR. This time we performed a touchdown PCR in order to become more specific results.
-Our three biobricks were purified by using a gel extraction kit. After this, we inserted our biobricks in pSB1C3. The next step is screening colonies by PCR to find plasmids with the right insert.
-We made our devices for the InterLab Measurement Study by using the Biobrick Assembly Method. After this we transformed electrocompetent E. cloni with our devices. First we grew our cells under the recommended conditions of iGEM on plates and afterwards we made a liquid culture. On Friday, we made our standard curve based on fluorescein and we measured our devices.
-We used the NEBuilder® HiFi DNA Assembly Master Mix to assemble our gBlocks. After doing a PCR, we visualised our results on a gel. We obtained a positive result for gBlocks 5 and 6. So we transformed these assembled gBlocks in electrocompetent E. cloni. We checked our transformed gBlocks on PCR, but a confirmation by restriction mapping is still needed.

-The transformation of chemocompetent cells for the Interlab Measurement Study was not efficient, therefore we repeat this with electrocompetent cells. We grew the transformed cells overnight and performed a miniprep.
-To check if the operon of ΔTarΔCheZ is OK, we used a PCR to confirm that all the genes are present.
-After performing a PCR, there were no bands visible of the assembled gBlocks, probably we didn’t use enough Gibson Assembly Master Mix
-We started making three BioBricks starting from our gBlocks. We performed a high fidelity tail PCR to include the prefix and suffix in our biobrick. The parts were digested and purified on a gel.
-We ordered materials for leucine and AHL detection

-Perform gel electrophoresis to confirm double knock-outs: 2 colonies of ΔTar ΔCheZ and 4 colonies of ΔTar ΔTsr still had ΔTar. This means we have our double mutants! We still need to check ΔTar ΔCheZ to be sure the rest of the operon is still intact.
-We performed a motility test to verify the phenotypical change of knocking out CheZ
-Assembly of gBlocks by using the Gibson Assembly Method
-We decided to participate at the iGEM 2015 Measurement Interlab Study. Therefore we transformed E. cloni with the biobricks J23101, I12504, J23106 and J23117.

-Making double knock-out strains by P1 transduction
-Performing PCR and gel electrophoresis to confirm that we have double knock-outs

-Performing a PCR and gel electrophoresis to confirm that the kanamycin cassette has successfully been removed from ΔTar and make a stock of this new strain.
-Preparations for phage P1 lysate for making the double knock-out strains

-Calculation of transformation efficiency of competent cells (E. cloni)
-Order 3 knock-out strains (ΔTar, ΔTsr and ΔCheZ) & prepare a stock
-Order Chromobacterium violaceum CV026 transposon mutant for use in AHL detection & prepare a stock
-Removing the kanamycin resistance gene of the ΔTar by transforming a plasmid with recombinase gene

-Prepare LB agar medium
-Prepare competent cells (E. cloni) and test competency