2023D3SEG23Wadidie

Determination of point mutation in the T7 RNA polymerase coding sequence of mutant #3 Draft Report 3 : Result and discussion Bolo Wadidie 300043378 BCH3356 Section E Groupe 23 TA: JACK November 2 nd , 2023 University Of Ottawa

Results To identify the specific point mutation present in mutant #3, the T7 RNA polymerase insert was integrated into the plasmid vector, pTAC-MAT-Tagl. This was achieved by amplifying the T7 RNA polymerase insert using PCR amplification and PCR purification. The results of the PCR reaction were analyzed by gel electrophoresis. As shown in Figure 1, bands were present for the purified and unpurified PCR product (wells #2 ,3 and #5), indicating a successful PCR reaction. The negative control channel had no bands (well #4 ,6), which was expected. Figure 1. Gel electrophoresis on 1% agarose gel results purified PCR product, the unpurified PCR product and the negative control of T7 RNA Polymerase. 10X loading buffer was added to the purified PCR product, unpurified PCR product and the PCR negative control. Well #1 contains 10 uL of MassRuler Express Forward DNA ladder (Thermofish). Well #2 and #3 #5 contains 10 uL of the purified ED,NT and unpurified ED NTamplicon, respectively. Well #4 and 6 contained the negative control. The gel electrophoresis was conducted at 100 volts for 40 minutes. The fragment lengths (in base pairs) were indicated alongside the DNA ladder on the left side (well #9). To facilitate the ligation of the insert and vector into the recombinant vector, both the insert and vector amplicons were altered to incorporate restriction sites. This enabled the separate digestion of the insert and vector using HindIII and EcoRI. The insert concentration was determined to be 20 ng/uL, while the vector concentration was calculated at 11.044 ng/uL. Following the completion of the digestion process, the insert and vector were combined through ligation, forming the recombinant plasmid. The competent KRX cells underwent transformation to harbor the T7/pTAC-MAT-Tagl recombinant plasmid. These transformed cells were plated on ampicillin-containing agar plates. Notably, observations

done the Amino acid comparison between our Mutant and wild type where Lysine(k) was substituted for a Leucine (L) at position 172 in the amino acid. T7RNA_M ATCATATGGCTCACAACCACCGTCACAAACACAAGCTTATGAACACGATTAACATC GCTA 120 T7RNA_Wt -----------------------------------------------------ATC ACTA 7 *** *** T7RNA_M AGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCA AC ACTC TGGCT G A CCATT 180 T7RNA_Wt GCA--------------------------------------A GC TCTC AGGC CT G GCATC 29 * * * *** *** *** T7RNA_M A CGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATG AGTCTTACGAGATGGGTG 240 T7RNA_Wt A TGG------------------------------------TG CATTTTAC---------- 43 * ** ** * **** T7RNA_M AAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACG 300 T7RNA_Wt ----------------------------------------------------------TG 45 * T7RNA_M CTG CCG CCAAGCCT CTC ATCACTA CCCT A CTCCCTAAGATGATTGCACGCATCAACGACT 360 T7RNA_Wt CTG AGG AGAAGGCT GCC GTCACTA GCCT G TGGAGCAAGATGA------------------ 87 T7RNA_M ACAA GG CTCGCAAGTCTCGCCGTATCAG CCT TGAGTTCATGCTTGAGCAAGCCAATAAGT 1320 T7RNA_Wt ---A TG TGGAAGA--------------G GCT GGAG------------------------- 105 * * * * ** *** Figure 3. Nucleotide-nucleotide alignment of mutant #2 and the T7 RNA Polymerase wild-type sequence, using Clustal Omega program. The mutation inserted in by technical staff is highlighted in but, at position 117 of the mutant sequence, where an Guanine was switched to a adenine .. There are so many mutations intentional point mutations are in position With bleu hightlights we got A to G and G to C and the unintional point mutations are green . Contig1 LN LRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSL 240 Wildtype LN KRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSL 229 ** ********************************************************* Figure 4. Protein-protein alignment of mutant #3 and the T7 RNA Polymerase wild-type sequence, using Clustal Omega program. The mutation inserted in by technical staff is highlighted in green, at position 172, where a leucine was switched for lysine . Discussions In the case of mutant #3 of the T7 RNA polymerase, initially, the exact location and type of point mutation within its coding sequence were unidentified. Utilizing recombinant DNA techniques, the T7 RNA polymerase mutant #3 was isolated and purified, leading to the identification of the point mutation: a substitution of leucine with lysine, precisely occurring at position 172 in the translated mutant sequence. Figure 1's data analysis indicated a successful PCR reaction due to observable bands in both purified and unpurified PCR products (wells #2, 3, and 5). While the negative control showed no bands (as expected), the band clarity was not optimal, likely due to

potential DNA loss during purification. The observed bands' concentration on the electrophoresis gel could be attributed to poor mixing of solutions during the process. An estimated recovery yield of 90-95% could have been achieved, aligning with the expected values from the manufacturer. Regarding transformation treatments, low colony numbers were observed across all treatments. Treatment I displayed only two colonies, indicating a lack of vector integration within the bacteria. Treatment II had a single cell, expected as there was no T7 amplicon. Treatment III showed minimal colony formation, as expected due to digestion with a single restriction enzyme (HindI). Treatments IV and V, designed as negative controls, yielded no colonies, aligning with expectations. Figure 2's data revealed positive results in Treatments I and III, with Treatment III displaying more visible bands, indicating its suitability for sequencing. The concentration obtained for sequencing exceeded the minimum requirement of 100 ng/uL. C= 163ng/uL. Further analysis involved using the Clustal Omega program for protein-protein alignment, confirming the Leucine to Lysine mutation at position 172 in the mutant sequence. This mutation was one of the five deliberately inserted mutations by the technical team into the T7 RNA polymerase coding sequences. While the identified mutation in mutant #3 was a Leucine to Lysine substitution at position 172, additional investigations are warranted to determine its impact on the T7 RNA polymerase function. Future analysis could reveal whether this mutation affects the enzyme's function, either causing a loss, gain, or no change in its functionality.