RESULTS
"The beginning of knowledge is the discovery of something we do not understand."
- Frank Herbert
"The beginning of knowledge is the discovery of something we do not understand."
- Frank Herbert
Since the beginning of our journey, one of our main motivations has been to go beyond theory and validate the functionality of our genetic designs in the laboratory. Through a process of constant effort and learning, and despite the difficulties encountered, we managed to make certain progress into our investigation.
Agarose gel electrophoresis of PCR-amplified scFv1-EpoR-N-mCer (1st lane, ~2700 bp) and scFv2-EpoR-C-mCer (2nd lane, ~2400 bp)
Both of the transcriptional units synthesized were successfully PCR amplified using a high-fidelity Q5 polymerase. Agarose gel electrophoresis revealed the presence of amplification and amplicons of the expected size: ~2700 (scFv1-EpoR-N-mCer) and ~2500 (scFv2-EpoR-C-mCer).
Future considerations:Downstream steps of the workflow were not addressed due to time limitations. Once obtained the TUs with the corresponding Bb prefixes and suffixes, the workflow can be continued as designed.
The initially proposed pre-cloning into pSEVA281 was not achieved. When an RFP dropout was used, a low effiency was observed in the ligation. The few potentially positive white colonies were picked and inoculated into liquid media. However, in two different sets of pre-cultures adding up to 9 tubes, no growth was observed after 24 hours on either of them. We hypothesized that there might be a problem of plasmid stability, which would compromise antibiotic resistance. We decided to utilize the CloneJET PCR Kit for efficient pre-cloning.
Replica plate elaborated for colony PCR screening of pJet1.2 transformants
Sequencing results from 3 clones of pJet1.2+VHH
The transcriptional unit from synthesis was successfully pre-cloned into pJET1.2. The screening of transformant colonies through colony PCR resulted in a great number of samples with amplification of the expected size ~1600. 4 of the colonies were pre-cultured for plasmid purification and 3 of the resulting plasmids were sequenced. The results of all the analyses permitted verification of clones 1 and 3, while the second presents an insertion mutation (T) at position 622, which falls at the nucleotide -60.
Despite the mutation not affecting the CDS, which would have resulted in a change in the reading frame, the mutation might cause a change in the promoter strength. Thereby, clones 1 and 3 are chosen for downstream experiments.
Colony PCR of pJet1.2+VHH (lanes 17-36)
The assembly of both fragments by Gibson Assembly was not achieved. In a first attempt, a very few transformants were visible, yet no colonies were observed in the positive control. Therefore, the result was discarded. A possible reason for such result could be the insufficient competence of the cells used. Therefore, we decided to repeat the experiment with commercial NEB® Stable competent cells. This time, more colonies were observed, both in the sample and positive control.
Screening of the transformants by colony PCR, however, led to no positive result, given by the absence of amplicons of the expected length (~ 3000 bp) in the samples, as well as no amplification in the positive control (pSEVA281+RFP).
Colony PCR of 36 colonies transformed with the product of IGG6 Gibson Assembly Reaction
Despite the inconclusive results from our last attempt of assembly, we believe that part of our troubles come from a reduced efficiency of the Gibson reaction. Due to the usage of the fragments from synthesis directly, which have a concentration of 10 ng/μL, with the volumes established in the kit’s protocol we are only able to achieve the suggested concentrations by minimum. Prior amplification of the fragments through PCR could help concentrate the DNA and ensure a high fragment-vector ratio, which is recommended for higher efficiency. This could also be achieved by increasing the total reaction volume. In addition, to reduce the noise originated from undigested plasmids, gel purification followed by clean up could help minimize the amount of undesired plasmids. Altogether, we expect these ideas to lead us to a positive result in order to continue the workflow.
The assembly of this cassette was decided not to be approached until a simpler Gibson Assembly reaction had been achieved. However, due to the requirement of greater amounts of pmol per fragment in a reaction involving 4-6 fragments, a previous PCR amplification appears compulsory to meet the required DNA concentrations.
Saccharomyces cereivisae growth in YPD with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: OD600 nm
RFP-expressing Escherichia coli growth in LB monitored through optical density, with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: OD600 nm
RFP-expressing Escherichia coli growth in LB monitored through fluorescence intensity, with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: fluorescence intensity
Based on the results from the growth inhibition assays, aimed to provide more evidence regarding AFB1 toxicity on E. coli and S. cerevisiae, we found that the effect was more apparent in the yeast species.
A slightly declining trend is observed in the growth rate of S. cerevisiae with increasing concentration of the toxin. However, no serious impairment is observed, supporting the use of this organism in the context of our project.
Meanwhile, E. coli growth does not seem to be signficantly affected under the experimental conditions tested, as revealed by both types of measurements. However, the assay performed does not provide information on the cell wall's permeability to AFB1. Thus, more experiments would be required to determine the viability of an E. coli-based receptor.
During the course of our project, various have been the changes of ideas and difficulties which have led us to a delayed start in the laboratory activities regarding our final design. Until that point, we managed to generate useful parts for future endeavours, including parts from the EMeRALD platform and for the construction of a novel shuttle vector.
Regarding our testing designs, we managed to complete the first steps of the proposed workflows. However, due to time limitations we could not see our project grow to its whole grandness during this iGEM season.
We understand that regardless of the passion and effort put into a scientific goal, research does not always turn out as expected. Nevertheless, we have the determination to keep working in this line and hope to find ways to keep up with this project.
