Mutagenesis Project Rundown

Mutating a Gene Essential to the Glycolysis Pathway in Deinococcus radiodurans

September 16th, 2022

Introduction to the Project

Deinococcus radiodurans ( abbreviated D. radiodurans), an extremophile bacterium capable of withstanding great amounts of UV radiation, is dependent upon crucial metabolic pathways which aid in its survival, a couple of which are the Pentose Phosphate Pathway (PPP) and the Glycolysis pathway. For the purposes of this project, the Glycolysis pathway is the one under evaluation, for it is responsible for regulating growth and development as well as helping the bacterium withstand oxidative stressors. D. radiodurans has a very essential enzyme in the glycolytic pathway, fructose 1, 6-bisphosphate aldolase (FBA), and this enzyme catalyzes the fourth step of glycolysis where "the six-carbon fructose 1, 6-bisphosphate (F1,6BP) [turns] into two triosephosphates: glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)" (Pirovich et al, 2021). The project aims to disable the fbaA gene which codes for the FBA enzyme by inserting a kanamycin resistance gene in the middle of the active fbaA gene. The goal of mutating the fbaA gene is to disable the FBA enzyme from functioning. The final procedure involves transforming the mutated gene back into D. radiodurans to monitor the glycolytic functions of the bacterium under oxidative stress. It is hypothesized that the absence of the FBA enzyme (due to its mutated gene) will cause the Glycolysis pathway to malfunction, leading to the possible death of D. radiodurans colonies grown under stressors.  

Methods and Instrumentation

To carry out the preceding project, methods and assays of gene mutation are necessary. First, the fbaA (about 1200 bp) gene was broken down into two parts, named the left flank and the right flank. Then, a 907 bp long kanamycin resistance gene was acquired, along with all the necessary forward and reverse primers of the three fragments (namely the left flank, the kanamycin resistance middle flank, and the right flank). To recall past experiments, multiple assays were used for the mutagenesis project to assemble the three fragments into one large hybrid fbaA fragment, including Gibson Assembly, HIFI assembly, and Overlap PCR. For context, the Gibson and HIFI assembly assays were used to assemble a different gene, and due the lack of success of these assays, the third assay was chosen to carry out the assembly of the hybrid fbaA gene which would include the kanamycin resistance gene insert. Overlap PCR, as the name implies, consists of two steps, a step where overlapping of the ends of fragments occurs without using any primers, and another where the amplification of the overlapped fragments occurs using the appropriate primers. This assay was first adopted by the student mentors Shawn Soares and Arnold Federico from a published research paper titled "Optimization of overlap extension PCR for efficient transgene construction" by Roland S. Hilgarth and Thomas M. Lanigan referenced below. Due to many failed attempts at producing a 3-fragment assembly, making a 2-fragment assembly was successfully demonstrated and achieved by the mentioned mentors. Thus, the acquired knowledge was passed down to the mentees, hence this project was developed. Overlap PCR is now being used in the laboratory to achieve the 3-fragment assembly. The project involves the frequent use of the Thermocycler for the PCR procedures as well as Agarose Gel Electrophoresis equipment to check for assembled bands corresponding to the correct size of the hybrid fragment. The first Overlap PCR procedure was attempted today, and the results will be documented through next week's blog. Stay tuned to find out the outcome of this mutagenesis project. 

References

Pirovich, David B et al. "Multifunctional Fructose 1, 6-Bisphosphate Aldolase as a Therapeutic Target." Frontiers in molecular biosciences. Vol. 8 719678. 11 Aug. 2021, doi: 10.3389/fmolb. 2021. 719678

Roland S. Hilgarth, Thomas M. Lanigan. "Optimization of overlap extension PCR for efficient transgene construction." MethodsX, Volume 7, 2020, 100759, ISSN 2215-0161,

https://doi.org/10.1016/j.mex.2019.12.001.

(https://www.sciencedirect.com/science/article/pii/S2215016119303358)