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

-Cloning our biobricks by use of the plasmid backbone originating from the InterLab Measurement Study. As a result,the biobricks CheZ-GFP, LuxI-His, LuxR-E and Ag43-YFP were obtained!
-Assembling the biobrick CheZ-GFP behind a strong promoter and RBS in order to characterize this biobrick.
-Assembling a strong promoter with GFP in order to compare the fluorescence to our library of promoters generated during the InterLab Measurement Study. Unfortunately the strong promoter was not green which indicates that the ligation did not work.
-Performing the OHHL quantification method and the leucine quantification method.

- Constructing the BioBricks LuxI-His, RBS-LuxI-His and CheZ-GFP, RBS-CheZ-GFP by high fidelity tail-PCR, digestion and ligation in pSB1C3
- Transforming these BioBricks in E. cloni, testing colonies by PCR and preparing for sequencing. The high fidelity PCR and cloning of other potential BioBricks was repeated.
- Making our strains ΔtarΔtsr and ΔtarΔcheZ electrocompetent
- Checking our colonies containing the assembled gBlocks by restriction mapping showed the presence of the original pUC19 vector in our samples. Treating the assembled gBlocks with DpnI should circumvent this since DpnI only cuts the methylated DNA.
- Digesting with DpnI eliminated the original pUC19 and not a single colony grew on the control plate transformed with plasmid only. Analysing the positive colonies showed presence of pUC19.

- Miniprepping and analyzing the assembled gBlocks is showing the assembled plasmid, but also the presence of pUC19 used as template. Transformation of the gel-purified correct plasmid was done in E. Cloni
- In parallel, assembling LuxI with gBlocks 1+2+3 and gBlocks 5+6 with gBlocks 1+2+3 was conducted with colonies that did not contain any pUC. Analysis of the gel was not showing any sign of digestion.

- Preparing electrocompetent cells
- Optimizing the tail PCR to make Biobricks
- Cloning the PCR products into pSB1C3, transforming the plasmids and screening of colonies to find the correct insert
- Transforming the devices for the Interlab Measurement Study
- Preparation of the standard curve based on fluorescein and measuring the fluorescence of the new devices
- Assembling the gBlocks using NEBuilder® HiFi DNA Assembly Master Mix and transforming the checked pcr products in electrocompetent E. cloni

- Switching from transformation of chemocompetent cells to electroporation due to low efficiency for the Interlab Measurement Study
- Checking the intactness of other genes in the tar-tap-cheRBYZ operon by PCR
- Ordering NEBuilder to repeat the failed Gibson Assembly
- Making three BioBricks starting from our gBlocks by tail PCR and restriction digestion
- Ordering materials for leucine and AHL detection

- Confirming the double knock-outs and checking the intactness
- Performing a motility test to verify the phenotypical change of knocking out cheZ
- Assembling the gBlocks using the Gibson Assembly Method
- Transforming E. cloni with the BioBricks J23101, I12504, J23106 and J23117 to participate in the iGEM 2015 Measurement Interlab Study.

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

- Checking the removal of the kanamycin cassette in the Δtar strain and make a stock of our successful knockout
- Preparing phage P1 lysate to make the double knock-out strains

- Calculating the transformation efficiency of competent E. cloni cells
- Ordering 3 knock-out strains (Δtar, Δtsr and ΔcheZ) & preparing a stock
- Ordering Chromobacterium violaceum CV026 transposon mutant for usage in AHL detection & preparing a stock
- Removing the kanamycin resistance gene of the Δtar strain by transforming a plasmid with recombinase gene

- Preparing LB agar medium
- Preparing competent cells (E. cloni) and testing their competency