Difference between revisions of "Team:Grenoble-Alpes/construction"

PROBE CONSTRUCTION

• PROJECT
  • BIOLOGY
    • BACTERIA CHOICE
    • TARGET SELECTION
    • PHAGE LYSIS & DNA EXTRACTION
    • PROBE CONSTRUCTION
    • CONSERVATION
  • ENGINEERING
  • DEMONSTRATE

CONSTRUCTION FOR PSEUDOMONAS AERUGINOSA

LYSIS

1. Plasmid backbone selection

Among all the backbones proposed by iGEM, we decided to choose one for our probe insertion. In fact, the 2017 IGEM Grenoble team showed by their experiments, that the pSB1C3-BBa_J04450 backbone was the most suitable for the detection of a red fluorescence.

The BBa_J04450 biobrick is composed of a RFP gene which is controlled by a LacI promoter inducible by IPTG. Moreover, it has a resistance gene for chloramphenicol to be able to select only bacteria that have been transformed.

Several teams showed that BBa_J04450 LacI promoter leaks. iGEM Grenoble 2017 highlights the role of IPTG, at least 180 minutes after induction. IPTG appeared to be important in the detection because it triggers a shorter rise of the fluorescence. As we know, it triggers the transcription of lac operon inducing the expression of the reporter gene.

So BBa_J04450 was chosen because this iGEM part enables users to produce mRFP1, a fluorophore which is an engineered mutant of red fluorescent protein from Discosoma striata. This reporter gene is LacI sensitive and can be induced with IPTG. This biobrick was chosen to be able to see fluorescence even if no IPTG was added or adding it to have a more rapid kinetic production.

2. Design of the probe detecting a fragment of P.A characterizing the lysis

The addition of the bioinformatically designed probe in pSB1C3-BBa_J04450 is done using the Gibson technic.

First of all, as a reminder, the DNA fragment to characterize the lysis is :

The probe has been imagined based on the system of Cork Ireland 2015 team and all the data furnished by New England Biolabs (NEB) website. It contains :

- Two restriction enzymes producing cohesives end, SphI and NgoMIV. Their goal is to remove the little sequence in between on the bottom strand. Thus creating a perfect complementarity with the target and linearizing the plasmid. But at the end we trust that using PCR linearization could reduce the background of uncut plasmid.

- Two nicking enzymes, Nt.BspQ1 and Nb.BsmI, i.e. enzymes that cut one strand of the double stranded DNA. Thereby, the top strand is removed, allowing the binding of the target.

3. Probe insertion in the psB1C3-BBa_J04450 plasmid - Creation of BBa_K2629000

3.1 Preparation of psB1C3-BBa_J04450

To be able to use the Gibson technic, we had to “prepare” the backbone we chose. To do so, we decided to linearize it by using primers for a PCR. The insertion is between the backbone prefix and the inducible promoter.

All the experiments were done by ordering sequences to Integrated DNA Technologies (IDT).