This is a short post that will cover a few sentences describing the next steps on the experiments that I will do in the summer.
1. During my last experiments (before a pause to write my thesis proposal) I was having a problem generating competent KW20 cells. It seemed that the problem originated when I over-dried my plated by incubating them more than 30 minutes at 42 degrees, which made colonies to accumulate within the borders of the agar plate. I need to prepare new plates and re-test the transformation frequency of some competent cells that I have in the -80 freezer.
2. I need to repeat to confidently find out how much periplasmic DNA I am recovering, as well as to decrease chromosomal contamination. For this experiment I will transform 1ug of sheared gDNA of 86-028NP Nal into 6kb and 0.25kb average size with 1ml of KW20 rec2::spec competent cells. Competent cells were pelleted and periplasmic DNA will be extracted. Extracted periplasmic DNA will be used as donor DNA in a transformation with KW20. In the same experiment I will do a standard curve transforming 6 quantities (0.01,0.1,1,10,100,1000 nanograms) of sheared gDNA of 86-028NP Nal and of KW20 rec2::spec with KW20 competent cells as well . Colonies growing in Nal plates were transformed from periplasmic DNA, while colonies growing in Spec were transformed from chromosomal contamination. I will do this experiment 3 times with biological replicates.
3. I will repeat the same experiment with C. jejuni 11168-O to quantify periplasmic recovery and chromosomal contamination. In this experiment I will use 6kb and 0.25kb 11168-O::kanR as donor DNA and 11168-O rec2::CmR (CJ1211).
4. I will quantify periplasmic extractions of points 3 and 4 using Picogreen. In order to sequence periplasmic extractions I need to make Illumina libraries. I need a minimum 1 ng of DNA in order to make illumina libraries using Nextera XT. Taking in account that before sequencing I will purify recovered DNA fragments using a automatized gel extraction robot I need to compensate for the amount of DNA that I might lose during the purification process. Ideally, I would like to have as much DNA recovered as possible, but at least 2 ng of DNA will be the minimum necessary to recovered to compensate up to 50% of DNA lost during purification (automatize gel extraction recovered between 50 – 80%).
5. I need to synthesize the fragments with degenerate region, and make uptake experiments with synthetic fragments in Campylobacter jejuni