Team:IOANNINA/Notebook

Notebook

Week 1 Week 2 Week 3 Week 4 Week 5 Next Steps References

Week 1 (10-16/9) : Preparation of plasmid pSB1C3

The first step was to get familiar with the experimental procedures and make our first transformation of the plasmid pSB1C3-RFP, provided in the distribution kit.

  • After plamid pSB1C3-RFP was dissolved from distribution kit we transformed the high efficiency competent E. coli DH5a strains, provided by NEB. Growth of the cells happened at agar plates ( + Chloramphenicol) overnight at 37°C, giving the next day succesful results. We chose specific colonies and made precultures of pSB1C3-RFP (Vfinal=3ml) to isolate the pSB1C3-RFP plasmid with the Monarch Plasmid Mini-prep Kit.
  • The plasmids were digested with EcoRI to check that the isolated plasmid was the correct one and once we were sure, we made glycerol stocks of the cultures that contained the plasmid.
  • By the end of the first week, we prepared everything needed for the next one, specifically agar plates and LB for bacteria cultures (LB-Agar + Kan, LB-Agar + Amp).

Week 2 (20-24/9) : Preparation of pOSIP-KO and pE-FLP plasmids

We received the two plasmids that we needed for the chromosomal integration of two of our genes according to the Clonetegration protocol by (Cui and Shearwin, 2017). The plasmids were pOSIP-KO and pE-FLP provided by Addgene and arrived as bacterial agar stabs.

  • We applied a streaking method using sterile toothpicks in order to isolate the bacteria containing the plasmids and plated them in agar plates containing the required antibiotics. The culture plates were incubated at 30°C, as at higher temperatures the integrase gene is expressed thus leading to integration of the plasmid sequence into the chromosome. This would make impossible the isolation of the plasmid for a cloning procedure.
  • The next day and after the bacteria had grown in the plate, we made precultures of selected colonies and proceeded to miniprep the next day to isolate the plasmids. To check the efficiency of the miniprep we did an electrophoresis which indicated that we had none to very small concentration of both plasmids. We repeated the miniprep procedure but the same results occured again.
  • This was an indication that something went wrong in some stage of the procedure. As this system is temperature-dependent, we supposed that there could have been some accidental arisal of the temperature in some stage, resulting in chromosomal integration before the plasmid isolation. For the next week, we decided to make a colony PCR as described by Cui and Shearwick in their clonetegration protocol, to check for this possibility.
  • Also, in this week we received the antibiotic deactivation module by IDT, which was resuspended and ready for use once the pOSIP-KO plasmid was ready.

Week 3 (27/9-1/10): Troubleshooting

  • Due to the minimal concentration of pOSIP-KO & pE-FLP plasmids, we proceeded with a colony PCR to check for possible chromosomal integration. This process did not give clear results, but indicated that our initial suggestion was right. We repeated the colony PCR and had a strong indication that the chromosomal integration had happened.
  • For this reason, and due to the pressure of time we decided to restreak the initial agarose stab, starting in the end of this week.

Week 4 (5-7/10) : Isolation and purification of pOSIP-KO plasmid

  • This week we went on restreaking the initial agarose stabs and repeating the process of plating, making precultures and minipreps. The electrophoresis that took place at the end of the week showed that we had successfully isolated and cleaned the desired plasmids. To make sure we had the correct plasmids, we digested a small amount of the plasmid with EcoRI to check for the length of the DNA sequence.
  • To proceed with the HiFi DNA Assembly by NEB, we needed to linearize our vector pOSIP-KO through EcoRI and PstI digestion. To make sure that the two enzymes were fully functional, we tested them in different enzyme buffers (as their initial ones didn’t match) and decided on which combination gave the best result.

Week 5 (11-15/10) : Assembly of pOSIP-KO/TetX2-EreB and Clontegration

  • We were now finally ready to assemble our first gene cassette into the pOSIP-KO plasmid vector.

First, we did a digestion reaction with 2.5 μg of pOSIP-KO with the restriction enzymes PstI and EcoRI. Due to some problems during the digestion procedure, we were not able to proceed with the assembly. The digestions were repeated the next day.

  • The digestion of the plasmid vector was repeated and as the electrophoresis that followed showed, the linearized plasmid was ready for the assembly reaction

    • NEB HiFi DNA Assembly reaction

    • After quantification of both the linearised plasmid and the insert (antibiotic deactivation module) we ended up using a ration of vector: insert of 1:2 and specifically 0.04 pmoles of insert and 0.02 pmoles of vector in the NEB Hifi DNA Assembly reaction.

    Transformation-Clonetegration

    • After the assembly, we transformed NEB high efficiency competent E.coli DH5a strains and plated for the control reaction ampicillin plates and for the assembled pOSIP-KO plasmid Kanamycin plates with a lower concentration of antibiotics than normally used (we used 20ul/ml).
    • In the 1 h incubation of the transformed cells at 37°C, the integrase was produced thus leading to the chromosomal integration of the plasmid that contained the deactivation module.
    • The next day we observed colonies that were growing in a slow rate, however after 2 days of incubation in 30°C, full size colonies were picked.
    • We performed a colony PCR that showed the success of our clonetegration.

      • This week we also received the pOSIP-KH plasmid vector that will be used for the chromosomal integration of the other modules of our project into the chromosome. However, due to lack of time, it is possible that we will not achieve this goal until the Wiki Freeze.

    Proposed Experiments for the next steps

    Even though we didn’t have the desired time to finish all our experiments, we have done a design of the next steps needed to have a full proof of concept of our lab design.

    Test of Deactivation Module

    As we have already completed the first chromosomal integration of the antibiotic deactivation module, next step would be testing its efficiency. For this, different amounts of tetracycline and erythromycin (macrolide) would be needed to check which concentration can be deactivated in an efficient manner.

    Test of kill switch

    To test the kill switch that was designed by IISER-Tirupati, a plasmid construct of pSB1C3, containing the kill switch gene cassette would be cloned by the NEB Hifi assembly protocol and transformed into DH5a competent cells, in the presence of arabinose. The bacterial OD would be measured to check if the bacteria can live while mf-lon gene is expressed. An extra plasmid containing only the gene of bpDNase I under the control of an arabinose inducible promoter could be used as a control.

    Final bacterial strain construction

    If the testing of the kill switch gives the desired results, we could proceed with the chromosomal integration of the bpDNase I, using a second plasmid, pOSIP-KH, which would allow the insertion of the gene cassette in a different position of the chromosomal DNA than the one already used (pOSIP-KO).The gene cassette of the bpDNase I could be assembled with the antibiotic sensor module and integrated into the chromosomal DNA as one sequence. Mf-lon gene would be cloned in a pSB1C3 plasmid in two copies, the first one being under the control of mphO operator and the other one under the control of tetO operator.

    Test of antibiotic sensor-Proof of concept

    Once this bacterial strain is constructed, different concentrations of tetracycline and erythromycin would be used to check the efficiency of the antibiotic sensor module. The results of this experiment would be counted as the bacterial OD. In case the sensor works, bacteria would live or self-destruct depending on the presence or absence of the antibiotics. This would give the final proof of concept of our design.