Week 22: "Exam stress"

Micro: For the month of June, our iGEM-work continued solely at TEK.

TEK: We kept experimenting with the best concentration to use for our NaF-tolerance experiments. This week, we used a stock concentration of 800mM NaF. We had to reheat our NaF-stock as it is sparingly soluble in this concentration. This week we also began to make freeze stocks of our ALE, as “checkpoints” in our experiments.

Week 23: "The Daily Groove"

TEK: The routine evolution protocol continued. We took turns going into the lab, allowing each other to also read up on our curriculum for exams.

Week 24: "Critical Thinking"

TEK: After some trial-and-error with our sodium fluoride-concentrations, we realized that 800mM was too concentrated, and also needed to be reheated each day to be properly dissolved. We adjusted our experiments to use a concentration of 320mM NaF. This week, we also compared the original Rosetta strain to our ALE strain. Please see the results on our Results page.

Week 25: "Re-evaluations"

TEK: The 96-well plate experiments did not yield the results that we had wished to see. The growth pattern of the bacteria was too irregular. Therefore, we decided to start another NaF-tolerance experiment series and run it parallel to the 96-well plate experiment. This time, our bacteria would be grown on LB-agar plates. We cast LB-agar plates with chloramphenicol (CML). Unfortunately, the bacteria had not grown on the plates. We realized that the ALE must have lost its CML-R plasmid because we did not use CML on the 96-well plates. Thus, we decided to start this experiment anew with the original Rosetta-strain – this time on LB-agar plates with CML (30 µg/mL). The mutant evolved from these experiments will be referred to as ALE2. We made plates ranging in concentrations from 0mM to 500mM NaF.

The way we cast the plates at this point was sub-optimal. We created one bottle of LB-agar and added CML. This bottle was reheated for each day we cast new plates – followed by the addition of a required amount of NaF. We quickly realized that this procedure would decrease the quality of the antibiotic. So, from here on out, we opted to, instead, reheat only the agar, and mix it with CML and NaF in 50mL Falcon-tubes for each.

Week 26: "The Grind Continues"

TEK: We continued to run the 96-well plate experiments alongside the new agar-experiments. At this point, the growth of our ALE2 was very irregular on the plates. One day, the ALE2 bacteria grew on a plate with a concentration of 280mM NaF, the next, we had to decrease the concentration to >130mM NaF to observe growth.

Because we ran out of our 320mM NaF stock solution to the 96-well plate experiments, we instead decided to use our 500mM NaF.

Week 27: “Two Steps Forward, One Step Back”

Micro: We started out the week by transforming the E. coli Top10-strain with ampicillin (Amp) resistance followed by miniprep of the plasmid. We also ran a high-fidelity PCR on each of our DNA-pieces which encoded our dehalogenases and ran part of the PCR-product on a gel, to verify our DNA-pieces. We then saved the rest for later use. This week, our first Gibson assembly of our backbone plasmid and insertion piece took place. We attempted to transform this into the original Rosetta-strain and examined our results using colony-PCR.

This first attempt did not yield any useful results, so we repeated this workflow a few times over the course of the week. Our 3^rd Gibson assembly was successful, so we purified our plasmid containing our insertion piece, and we could now attempt to insert our dehalogenases into our backbone plasmid with Golden Gate (GG) Cloning.

TEK: NaF-tolerance-development of ALE2 on agar-plates continued.

Week 28: “New protocols and Reevaluations”

Micro: We attempted the first GGC this week. None were successful, despite us adjusting DNA-concentrations and tweaking the protocol. We also wanted to test if our fluoride-sensitive riboswitch worked, so we plated out ALE1 with no plasmid, ALE1 containing the pET51b plasmid with RFP and the variants with genes encoding the DeHa’s. The agar plates contained different concentrations of NaF and kanamycin. Some also contained IPTG and ampicillin as well. We did this to discern if the kanamycin-resistance of ALE1 corresponded with the amount of NaF that was in the plates. Unfortunately, cells were kanamycin-resistant regardless of whether the plate contained NaF or not. This pointed to a leaky riboswitch. This meant we had a lot of reevaluations to do. Therefore, we designed four new DNA-parts to test out and ordered them.

TEK: This week, we decided to see if our 96-well experiments had been effective, by testing whether our ALE-strain grew better than the original strain in NaF. We made overnight cultures of every other freeze stock we had made and measured the OD over the next few days. The resulting graphs did not look as we expected them to, partially because some of the wells had dried out, so we decided to try again later. We had also forgotten to add CML to the wells.

Week 29: “Ups and downs”

Micro: After all the failed GG-reactions, we thought it would be a good idea to test the working stocks of our primers for contamination. We found that one of them was, indeed, contaminated, so we threw it out. We also decided to modify our GG-protocol so that rather than a “single-tube-reaction”, we separated the reaction into cutting of the DNA-pieces and ligation of the cut pieces, respectively. This time, the reaction worked! Now, we had a sample with, presumably, our backbone plasmid with one of our dehalogenases, DeHa2, inserted!

TEK: With our second attempt at comparing the OD of our ALE1 and original Rosetta-strain, we added CML to the wells of the 96-well plate and stored it in a tightly sealed bag to prevent the wells from drying out. The OD was measured, but a lot of the bacteria had accumulated at the bottom of the wells. Therefore, the plate was stored in the refrigerator overnight, and the next day, the bacteria were resuspended, and the OD was measured again.

We also wanted to check the NaF-tolerance of the ALE2-strain. To do this, we made a dilution row of ALE2 and the original Rosetta-strain, respectively. We plated them out on agar-plates with varying NaF-concentrations. We found that ALE2 was more tolerant of NaF than the original strain!

Week 30: “Back to the Drawing Board!”

Micro: This week was spent waiting for sequencing results from various DNA-pieces. Among those were the plasmid backbone + insertion piece from our successful “Gibson assembly 3” and our newly ligated plasmid backbone + DeHa2, both with our forward and reverse primer.

We also decided to redo the test of our riboswitch, this time more thoroughly. In summary, these results revealed that our kanamycin resistance was constitutively active. This meant that we had to re-design our plasmid. For more details on these results, please click here.

TEK: With the newly acquired evidence that our ALE2-strain experiments were effective, we decided to keep evolving them to see how much we could push the NaF-tolerance of the strain.

Week 31: “Sequences and Successes”

Micro: For the first time, we had DNA-samples sequenced! The lucky winner of this honor was the DNA-sequence for our DeHa2. Unfortunately, though, the sequencing results were rather disappointing, and seemed to show a frame-shift mutation at the end of the DNA-product. However, we received a new and different miniprep kit, which yielded much higher DNA-concentrations than our previous one.

Lastly, over the weekend, we decided to test how much PFOA our cells could tolerate, by plating them out on agar plates with increasing PFOA-concentrations.

TEK: At this point, the ALE2-strain seemed to have reached a plateau in their NaF-tolerance. Furthermore, we had to focus all efforts on our main project, thus concluding the evolution of NaF-tolerance in E. coli Rosetta. This also concluded our work at TEK.

Week 32: “New... group members?”

Micro: Our group decided that it was rather difficult to keep track of all our new DNA-parts. None of them really rolled off the tongue. Therefore, we decided to name them. Introducing the newest (and arguably most important) wetlab-personalities: Anders, Ken, Ginny, and George! All but George had now successfully been cloned into our plasmid.

The rest of the week was spent GGC dehalogenases into our plasmids, running colony-PCRs and checking that everything was assembled correctly.

Week 33: “The battle of plasmids begins.”

Micro: Now that most of our plasmids were loaded with a DeHa-gene, came the time for our selection process. We began testing Anders, Ken and Ginny for both RFP and GFP-expression. Poor George was lagging behind a little, and still needed to be transformed into Rosetta.

Fluorescence microscopy revealed that our plasmid, Anders, seemed to have the clearest response to increasing NaF-concentrations.

Our supervisor, Lin, proposed that we should test Anders in both our NEB-cells and ALE1-strain. She theorized that the NaF-tolerance might impact the fluoride-sensitivity of the cells, so, once again we prepared our cells for fluorescence microscopy. Unfortunately, it seemed that Lin was right. This led us to wonder whether the same might be the case if we were to put Anders in ALE2. For good measure, we also wanted to see the fluorescence of Anders in the original Rosetta-strain, too. Thus, we started the process of transforming Anders into these two strains.

Week 34: “Choosing our plasmid”

Micro: We decided to test the riboswitch in Anders and Ginny respectively, as they contained two different ones. After reviewing the results, we settled on Anders as our final plasmid, as it fluoresced more consistently under different circumstances. Later, we sent in Anders to sequencing, both without and with various DeHa’s.

Over the years, many iGEM-teams at SDU have battled hard and bravely in numerous attempts to make calcium chloride competent cell stocks. Despite the words of warning from our supervisors, we decided to challenge history – and with great success! We made a batch of approximately 100 1mL tubes of competent Rosetta cells! Subsequently, we transformed Anders with DeHa 1, 2, 4 and 5, respectively, into the newly competent cells, followed by a colony-PCR. To conclude the week, we transformed Anders with DeHa’s into ALE2. We finished with a colony-PCR of these cells and made freeze stocks for future use.

Week 35: “Fresher’s Flu and Access Quarrels”

Micro: This week was rather challenging, with many people falling ill with fresher’s flu and security systems resetting for the start of the new semester. This meant that we had to retake lab safety courses for TEK, and therefore had a bit of a setback, timewise. This experience was especially sour, as we discovered this on a weekend after spending a whole day preparing samples and solutions for an experiment regarding optimization of IPTG-induction.

We were also seriously starting to investigate buying a fluoride probe to measure enzyme kinetics.

Week 36: “FACS, fluoride probes, and plan B’s”

Micro: At this point we got into contact with sales employees, but we feared that shipping times might put a stick in the wheel of our project. This was definitely the time to think about plan B’s.

Speaking of plan B’s… as we were preparing to transform our error-prone dehalogenases, we found that Rosetta had a difficult time stomaching our plasmid. This, combined with previous results that indicated that the riboswitch might work better in other cells, sparked the idea that maybe it was time to try a new bacterial strain. But would we even have time for that…?

On a lighter note, we got to try our hand at FACS this week at Odense University hospital, because it was difficult to determine the most fluorescent colonies at the UV-Vis-plate at TEK. FACS seemed to be a better alternative for sorting cells based on fluorescence.

Week 37: “Big decisions and Stubborn Strains”

Micro: We decided to order a new strain after all. We went with T7 Express Competent E. coli (High Efficiency), referred to as “NEB T7”, from New England Biolabs. While waiting, we utilized time in the lab as well as we could. We spent time optimizing our Golden Gate protocol on another strain, BL-21, while we waited. What a workload! This was near impossible because BL-21 DID. NOT. WANT. TO. GROW!

Weeek 38: "A welcome arrival"

Micro: Finally, our new cell strain, NEB T7, arrived! This meant go-time! Luckily, these cells were receptive to our plasmid, and willing to grow for us! This meant that we were finally able to run induction experiments – we wanted to verify that our Dehalogenases were expressed, before lysing the cells. The SDS gods were against us, however, so we could not conclude anything from the gels that we ran.

Week: 39: “Feelin’ the pressure”

Micro: This week, we transformed our error-prone dehalogenases into the NEB T7 strain and started running FACS on them!

Still feeling a bit down from last week’s SDS-page results, we decided that a more practical approach was necessary. We had to French press our cells and purify our protein to check whether they were induced. Despite leaky equipment and low protein yields, the SDS-pages we ran, confirmed that DeHa4 and DeHa5 were indeed induced.

As a side note, we suspect that DeHa1 and DeHa2 were also induced, but might be in the solid phase of the protein purification kit. Due to time pressure, we decided to only proceed with DeHa4 and DeHa5. Unfortunately, none of the departments at our university had a fluoride probe, which we needed to determine enzyme kinetics. So, we ordered one!

Week 40: “iGEM projects can only be 80% done”

Micro: This was a big week, as we selected for the best error-prone dehalogenases! This involved a whole-day intensive laboratory experiment, but oh man, did we feel accomplished!

Our work was not yet done, though; we also had to send in the 52 samples that we collected, to sequencing, so we would not end up testing duplicates in the end. Having the DNA-sequence would also allow us to predict the tertiary protein structures. Furthermore, we received the results from our whole-genome sequencing of ALE1 and ALE2 strains. Now we could finally deduce where the increased fluoride tolerance came from!