Week: 26.06 - 02.07
After an introduction into the lab and the different equipment, the first vaginal fluid simulant (VFS) was mixed and the pH got adjusted to 4.5 and 6.5.. After autoclaving the medium still contained some debris and changed color.
Week: 26.06 - 02.07
After familiarization with the golden gate assembly methods, we started designing the plasmids for the quorum sensing system (QS). The QS component parts were found in the iGEM part registry, and the sgRNA parts were designed based on gene target suggestions from dry lab. A more efficient lactate dehydrogenase (from Streptococcus bovis) was chosen to increase lactate output.
Week: 03.07 - 09.07
Inoculation of the VFS with E.coli and L. crispatus. Since the bacteria didn't grow, the VFS was prepared new and the pH got adjusted. First experiments with E.coli and L. crispatus with both pH and aerobic and anaerobic conditions.
Week: 03.07 - 09.07
All the parts for the QS were ordered from IDT and we continuously started with the design of the dCas9 system.
Week: 10.07 - 16.07
Further experiments were performed to find the best agar plate for isolated colonies.
Week: 10.07 - 16.07
The sgRNA were designed with the online tool chopchop and the RBS were designed with Sally's LAB RBS calculator.
Week: 17.07 - 23.07
A new medium simulating vaginal fluid (MSVF) was tested but after autoclaving there were some solid parts inside.
Week: 17.07 - 23.07
Transformation of level 0 parts and backbones from the iGEM kit and preparing glycerol stocks. Trying out different versions of mini prep to increase yield and measuring the yield with Nanodrop and Qubit-Kit.
Week: 24.07 - 30.07
The IDT parts for the QS arrived and were diluted with nuclease free water to have a stock solution and a working solution.
The first assembly of four different Level 1 plasmids was mixed, followed by the short incubation protocol. After the transformation only two assemblies worked. Therefore, the parts were mixed again and then incubated with the long incubation protocol overnight. Transformation, overnight culture, and mini prep of Level 1 and sending for sequencing, with unfortunately only one positive result. Design and ordering adapters for Level 2 assembly of the QS. The design of the dCas9 system was done and the parts were ordered from IDT.
Week: 31.07 - 06.08
The medium simulating vaginal fluid (MSVF) was tested again with success. Growth experiments were performed with different pH and air conditions.
Week: 31.07 - 06.08
Trying first Level 2 assembly. Preparing E.coli MG1655 competent cells and transforming further iGEM kit parts. Competent cells containing the plasmid pfd152, which contains the dCas9 enzyme, were made and aliquoted for further use.
Week: 07.08 - 13.08
Growth curves experiments were being planned. An overnight culture will be subcultured in the morning and then every two hours the OD will be measured. For platting the bacteria, a dilution series in H20: Peptone will be made and then 10ul is placed on the plate.
Week: 07.08 - 13.08
First positive sequencing results and further trying Level 1 assembly since not all of them worked. After not having success with the transformation of the backbone jump47-ts for weeks, it was being realized that the incubation temperature should have been 30°C and not 37°C. After switching the temperature, the transformation worked but the yield after the mini prep was still too low, even with trying a different protocol for the overnight culture.
Week: 14.08 - 20.08
MRS and LAPTg media, as well as LAPTg plates were prepared. The MRS media was being tested. Further preparations for the growth curves were done and a first test run.
Week: 14.08 - 20.08
Since there were still two level 1 plasmid missing a 2:1 assembly ration of the inserts and backbone was being tested, and the promoter was switched to a different Anderson promoter. The assembly with a new promoter and 1:1 ration worked out great and the assembly was confirmed with a positive sequencing result. PCR and gel purification for changing the overhangs of LDH for chromosomal integration with pOSIP-TT, amplification of the dCas9 out of the pfd152 plasmid and homology arms out of the bacterial genome.
Week: 21.08 - 27.08
First growth curves experiment with E.coli and L.crispatus in LAPTg and LB liquid medium and a dilution series for plating on both plates.
Week: 21.08 - 27.08
Since only the PCR worked for one pair of the homology arms, several different versions for the PCR and different agarose percentage for the gel were tested. In the meantime, the parts from IDT arrived. With all the sgRNA we did a PCR to anneal the promoter and change the overhangs. Unfortunately, we realized that the PCR fragments were too small to do Gel purification with the kit.
Trying Level 2 QS assembly and upstream assembly in the jump27-ts backbone without success. Transformation of both pOSIP-plasmids from the kit but only pOSIP-CH worked.
Week: 29.08 - 03.09
Ordering new sgRNA fragments where the promoter and scaffold is already attached and dCas9 enzyme with a degradation tag from the Twist. In the meantime, we tried Ethanol purification for PCR sgRNA products. It worked for one sample, but the sequencing showed many mutations in the gRNA.
Trying Level 2 assembly without success.
Week: 04.09 - 10.09
Arrival of new sgRNA and assembly with great success. Making agar plates for blue- white screen and trying screen. No colonies grew.
Week: 11.09 - 17.09
Growth curves with L. gasserei and L. crispatus in MRS.
Week: 11.09 - 17.09
PCR with new design primers for LDH assembly to change the overhang for pOSIP- CH chromosomal integration. After the sequencing we tried to integrate it, which did not work out. The five different RBS level 1 plasmids were assembled and transformed.
Week: 18.09 - 24.09
Growth curves with L. crispatus and E.coli in co-culture. An overnight culture for each of the species and one containing both was made, and the next day the same procedure as for all growth curves experiments. Each culture was being platted on COS and LB after diluting in H20: Peptone.
Week: 18.09 - 24.09
After weeks of unsuccessful Level 2 assembly for the QS, we realized that the backbones in the Level 1 assembly were wrong. The Level 1 assembly was mixed new, transformed, followed by a positive sequencing result. Agar plates for the blue white screen were prepared by the media kitchen. Trying a different version to plate the LacZ sgRNA in the pfd152 competent cells. No success in inducing the expression of the dCas9. Only blue colonies.
Week: 25.09 - 01.10
With the new Level 1 assembly for the QS, we started with the Level 2 assembly. Preparation of RhlI level 1 plasmid to test with metabolomics. The sgRNA were transformed in the competent cells containing the dCas9. Overnight cultures were inoculated, and the first measurements with the D-lactic kit were made.
Week: 02.10 - 08.10
Visualization of growth curves.
Week: 02.10 - 08.10
Since the first try for the Level 2 QS plasmids was unsuccessful, the experiment was being performed again. This time with success. Further measurements of the efficiency of the sgRNA and first measurements of the RhlR + CFP assembly and the RhlI Level 1. Also testing the RhlI samples.
Week: 9.10 - 12.10
First measurement of the lactate dehydrogenase from the Streptococcus bovis in the knockout strain. Testing the expression of the LDH with constitutive promoter expression, inducing it manually by adding C4-Hsl to the RhlR + LDH Level 2. Testing the self-induction of the Level 2 RhlR + CFP construct with co-transformation of RhlI, which produces the auto-inducer molecule.