Throughout recent years in molecular biology there has been an interest in studying the effects of the transformation of enzymes to allow for heightened, improved, or altered abilities. In what was only a mere 50 or so years ago in 1953 when Francis Crick, James Watson, and Rosalind Franklin discovered the structure behind DNA (Elkin, 2003). Later on the theory of transcription and translation was proposed by Francis Crick, and later was expanded in 1961 to include the idea of amino acids being used to specify for certain codons that would then leads to proteins (Crick, 1970). This was the building block to the rest of molecular studies, and began the research towards the altering DNA, proteins, and other enzymes, for improved or different uses such as Genetically Modified Organisms (GMOs) to create sustainable food resources (Gepts, 2002), to knock out genes for research purposes, and to cure diseases through understanding how to alter how certain genes express (Horton, 2003).
This idea of RNA being the script to the expression of genes is what leads to the interest in altering RNA polymerases through the ligation of other polymerases into the sequence. This has led to the hypothesis that through the ligation of a recombinant plasmid vector, with an altered coding sequence, into a suitable plasmid vector, with the help of PCR amplification and sub-cloning techniques, the altered gene can have its expression, purity, and sequences. In this study, the focus is on the ligation of a coding sequence of our individual T7 RNA polymerase into a suitable plasmid vectors for the production of the protein pTAC-MAT-Tag1. T7 RNA polymerase was discovered in the 1970’s and has since be used as a tool throughout molecular biology due to is versatile roles in prokaryotic, eukaryotic, and even cell free systems (Wang et al, 2018). Another role of T7 RNA polymerase has recently been discovered, as it has been proven to be a strong transcription system in synthetic biology and allowing us to redesign and fabricate the biological systems we need that are not currently in the world (Polizzi, 2013).
T7 RNA polymerase under mutagenic conditions has already been shown to be able to express programmed mutation in specific regions under engineered retrons (Simon, Morrow, and Ellington, 2018). This is found to allow the production and evolution of already existing genes to diversify and to allow the creation of environmentally-selected antibiotic resistance genes Simon, Morrow, and Ellington, 2018. It now has mutation rates occurring in the targeted regions, happening about 37 times faster than the compared background cellular mutations, allowing of the phenotypes to continue to self-evolve Simon, Morrow, and Ellington, 2018. The T7 RNA polymerase is used specifically in this experiment due to its ability to synthesize RNA from any point in DNA as long as this point is located downstream of the promoter, given that T7 RNA having a promoter of around 20 base pairs (Poitras, 2018). T7 RNA polymerase can effectively synthesis modified RNA due to studies which found that T7 RNA polymerase increases the creation of mutated RNA along with mutated ATP allowing even highly mutated strands to be synthesized (Milisavljevič et al, 2018).
In this lab we are using point mutated modified strands which is why T7 RNA polymerase is a strong choice. Specifically, we insert our specific recombinant T7 RNA polymerase into pTAC-MAT-Tag1 to determine what point mutation has taken place in the particular T7 RNA polymerase. The goal of the experiment is to determine whether the point mutation is glutamine for an alanine, serine for an alanine, or lysine to leucine. This is done through the ligation of T7 RNA polymerase; this sample is then amplified.
In conclusion throughout this study we ligate, amplify, analyze, and record a variety of different point mutations in T7 RNA polymerase. We use this information to analyze and record how these point mutations effect the expression of pTAC-MAT-Tag1 based on the different point mutated strands of T7 RNA polymerase. This allows us to see if modified, the strands can gain abilities and resistances to substances such as ampicillin which originally would have inhibited them.