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

-We are constructing the BioBricks LuxI-His, RBS-LuxI-His, cheZ-GFP, RBS-cheZ-GFP, RBS-cheZ-GFP-RBS-PenI-Term-PpenI-RBS-RFP-Term by high fidelity tail-PCRn digestion and ligation in pSB1C3. We transformed these BioBricks in E. cloni and tested these colonies by PCR. The BioBrick RBS-cheZ-GFP-RBS-PenI-Term-PpenI-RBS-RFP-Term was confirmed by PCR and prepared for sequencing. The high fidelity PCR and cloning of other potential BioBricks was repeated.
-This week, we made our strains ΔtarΔtsr and ΔtarΔcheZ electrocompetent.
-Further, we would like to characterise a BioBrick. Therefore, we pasted RBS-cheZ-GFP after the promoter J23101. The idea is to test this BioBrick in a cheZ knock-out strain and to prove the presence of GFP.
-Our colonies containing the assembled gBlocks were checked by restriction mapping. The presence of the pUC vector in our samples made the restriction mapping complicated. This is why we chose to also investigate different samples who were treated with DpnI after the Gibson Assembly. The DpnI only cuts methylated DNA and thus only the original pUC. Unfortunately, the restriction mapping did not give the expected result.
-To have a backup plan, we also repeated the Gibson Assembly with pUC and the enzyme DpnI. After cloning and restriction mapping, we concluded that our sample contains only pUC.
-Our fourth gBlock is more or less 600 bp. To increase the amount and concentration of this gBlock, we performed a Phusion high fidelity PCR.

- We obtained assembled gBlocks 1-2-3 and 5-6 in mini prepped form. On gel, we discovered that we have assembled gBlocks in pUC, but also a band at the height of pUC. We cut out the correct band, did a gel purification and transformed again.
-In parallel, we further tried to assemble gBlocks 1+2+3 with gBlock 4 by using two colonies who did not contain pUC. Later, we noticed by PCR that the assembly was not correct.
-We also tried to assemble gBlocks 1+2+3 with 5+6, this would give us the total plasmid for cell B. On gel, we saw that the restriction did not work. Probably, there was a problem with the restriction enzyme.

- 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