An Overview of the Projects for Spring 2024

 Introduction:

    The current semester will include a continuation of previous projects as well as the introduction of new experiments which will be overviewed in this week's blog post. From the previous year the projects to be furthered include the development of a qPCR method to test temperature effects on CRISPR-Cas proteins in Deinococcus, DNA extractions for sequencing attempts, and utilizing the 7kb plasmid from Deinococcus aquaticus. New projects will include engineering other native Deinococcus plasmids, initially the 5kb found in Deinococcus ficus, and the analysis of newly sequences genomes to ideally find a target to genetically engineer Deinococcus caeni, and learn more about the structure of Deinococcus sonorensis. Below is a detailed explanation and background of each experiment and where each stands at the present time. 

The qPCR experiment to test the interaction of temperature on the transcriptional regulation of CRISPR-Cas proteins has been adjusted from previous semesters. Before it was to be tested on D. geothermalis, but given that this species has an large range of temperatures which it can survive at and that is has more than one CRISPR-Cas system in its genome, the project has switched to D. aquaticus. The benefits aquaticus has is not only a textbook Type IIIA system with no repeated proteins in its sequence, but a standard 30 degrees Celsius growth temperature and hopefully an easier specie to test for heat and cold shock parameters. It was realized previously that the temperatures, high and low, must be verified to induce a shock response from the cell in order to solidify results later on if any effects are seen on the regulation of cas proteins. Therefore two genes have been selected, a heat shock and a cold shock protein, in which primers have been made and will be validated to confirm a temperature range that will be adequate for protein testing. The cas proteins to be tested will also be Cas1 and Cas10, those primers set to be validated next week. 

The DNA extractions for sequencing have been mildly adjusted according to new requirements and abilities of updated technology. The first sequence to be completed will be D. caeni, especially important given that is has just recently been proven transformable using plasmid Prad1, and was isolated this week. The initial OD600 value was 0.23, and 10ml of culture was compounded and put through the DNeasy kit with the following modifications. Two washes of CB solution were done rather than one, bead beating was conducted for 3 total mins (1 min on/1 min off on ice) at max speed 5 m/s, and 25 ul of PCR water was used for elution and allowed to incubate at room temp for five minutes prior to centrifugation. The results from this extraction was 19 ul of sample at 650 ng/ul with a 260/280 of 1.96 and 260/230 of 2.14. D. sonorensis was grown from freezeback this week and will undergo a similar extraction with increased bead beating time to account for its difficult biofilm structure. 

The 7kb plasmid has been isolated prior, but the isolation has not been optimized for a significant amount of time with very little progress despite best efforts. The project has then shifted to a genetic engineering angle, hopefully by isolating the 7kb by long amplification PCR as opposed to straight from cell culture. The Gibson assembly will be used, first by amplifying the chloramphenicol gene from Prad1 with a specific set of primers with extended overhang regions complementary to the overhangs designed for the long amplification of the 7kb plasmid. The isolation of CMr has been successful, and the 7kb was unsuccessful the first attempt. A further test will be conducted comparing the results from colony PCR and previously isolated plasmid, as well as using the M23 gene of the 7kb as a positive control for each type of template. Once this has been accomplished assembly will be attempted and the 7 kb will be reintroduced to many other Deinococcus species, including D. caeni. D. aquaticus, and D. geothermalis. If the long amplification process can be verified, an identical mission will be attempted with the native plasmid from D. ficus, which would be suitable for even more engineering with its smaller size. 

Finally, from the sequence of D. caeni there will be a bioinformatic search for an ideal knockout gene, which will produce a phenotypic change to be evaluated. The transformation of D. caeni was recently verified with Prad1, and thus this plasmid will likely be engineered to induce the knockout of the specified gene and form proof of concept for even further experiments. The data has not been fully acquired yet so as soon it is available the gene of interest will be determined. 

The descriptions above are not a complete insight and will be expanded upon as work is accomplished for each one individually. Preliminary work has begun, but the goals for next week include primer validation of two sets of primers, DNA extraction of D, sonorensis, the long amplification of the 7kb, and hopefully a sequencing attempt of D. caeni. 



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