As already mentioned, our three attempts to transform the e coli cells with our 2019-2020 constructs did not work. In the first attempt, the main problem we had was that the ampicillin was expired, so the cells were not selected properly. After buying new ampicillin from a local provider, we made a second bacterial transformation. However, the bacteria did not grow in the LB+AMP plates. For this reason, we made a gel electrophoresis to find out what the problem was and looked for the IDT receipts, realizing that we had confused linear DNA with plasmid. Finally, with the last competent cells we had, we made a third transformation with the plasmids of constructs 1, 2, 3 and 6. However, there was still no growth in the antibiotic.
Despite not having obtained satisfactory results for the progress of our project, we were able to learn a lot about laboratory protocols and the necessary perseverance to work in a scientific environment. Likewise, we were able to work on the research so we have more knowledge about the next steps to make our solution a reality.
Once we confirm the transformation of cells with the plasmid constructs, we are planning to test if the DNA displays RFP by doing what we did back in September 17th 2022: adding transformed bacteria in different concentrations of cadmium to see any different shades of red produced by different concentrations of produced RFP.
Once confirmed that the transformation has been completed successfully and there are no issues regarding the expression of RFP, our group will proceed to the next phase of our investigation. This would consist of obtaining data concerning how well our plasmid and cell-free system design works within its intended objective. For this we plan to dry freeze the cell free system with our plasmids into dipstick-like devices, similar to those found in water PH testing kits. For which we would later evaluate our design's success or failure rate with genuine soil. We expect to accumulate diverse data when exposing our plasmids to contaminated and non-contaminated soil. From this, we will conclude from the previously mentioned data to either modify or analyze specific features of our methodology or our tested variables. This section would also include testing the sensitivity of the plasmids, ensuring that they give both accurate and precise readings of the data they are meant to report.
Rhizoremediation uses “mutual interaction of plant roots and suitable microbial species” (Verma et al.) in the rhizosphere and is considered the most effective, ecological method of biodegration and bioremediation of contaminants (like Cadmium). Our project strives to reach this point where we can suggest the usage our bioengineered bacteria for Cadmium remediation in cacao plantations.
However, through further research, we also found that the plant-microbe relationship may also further promote the efficiency of phytoremediation. Phytoremediation is a method used to remediate contaminants using accumulator/hyperaccumulator plants, which are plants that can “accumulate metals at high concentrations” at parts that can be harvested and later incinerated. (Verma et al.) However, even hyperaccumulator plants may not function optimally beyond certain levels of toxicity. Hence, plant growth promoting rhizobacteria (PGPR), which are mircroorganisms that live “in and around the roots” (Verma et al.) of plants, are implemented to reduce stress and support the plant through numerous mechanisms, including accumulation, transformation, sequestration, and degradation of heavy metals.
DNA. Due to strict customs regulations in Peru, it is expected that there will be difficulties in importing redesigned DNA constructs from IDT and Twist.
Antibiotics (laboratorial). In Peru, we only have access to pharmaceutical antibiotics which may not be suitable for our experiment because it may be too strong or may not be pure anitbiotics. Hence, we also have to import laboratory antibiotics from the US which takes 60-90 days.
Competent cells. In Peru we only have access to limited supply of competent cells from a local university. It would take about 60-90 days to import competent cells from abroad.
Hence, mostly concerning time, it is optimal that we create as few errors in our experiments as possible for these essential ingredients are difficult to obtain.
We discovered that we were using linearized DNA (Which has to be cloned before transformation) at the very end, meaning that all of our attempts of transformation undoubtingly failed and thus our ‘transformed’ products did not show any signs of antibiotic resistance nor RFP.