Our first experiment consisted of adding 8 5’ UTRs to the plasmid vector of E. coli strains DH5α and BL21(DE3). Our vector consisted of a promoter (J23100) and CAM resistance sequence. First, we hybridized the UTR constructs we received from the vendor. This process was followed by the extraction of plasmids. We then digested the extracted vectors using enzymes EcoRI and XbAI. We added our UTR sequence, which SpEI and PsTI digested, and ligated the mixture. This mixture was then purified using agarose gel of 1% concentration which confirmed the completion of the ligation. The purified mixture containing the 5’ UTR along with the reporter gene was then transformed in E. coli followed by extraction. The transformed cells were then streaked and incubated. A similar process was repeated downstream of the UTR sequence to add the reporter gene using suitable enzymes. After the final transformation, we streaked the E. coli with a modified plasmid and observed the red glow of red fluorescent protein in 6 constructs. These 8 colonies were then streaked on a master plate and the colonies grown were analysed using spectrometry.
The results of our first 8 UTR constructs served as a basis for the dry-lab work and enabled further simulation that we used to order our next set of 11 UTRs. This time, we decided to optimize our process and ordered a construct which had both promoter + UTR which we added to a plasmid containing red fluorescence protein. This saved us a lot of time. This time the plasmid vector used contained only the antibiotic resistance and the RFP sequence. We first extracted this plasmid from the E. coli strain and digested it using EcoRI and XbAI. To this, our promoter + UTR construct was added after digestion with SpEI and PsTI. The mix was then ligated and purified using agarose gel of 1% concentration which indicated a successful ligation. This recombinant vector was then transformed into E. coli and streaked. The presence of a red glow in all 11 of these sequences indicated a successful experiment. These red colonies were then streaked on a master plate, and colonies were grown. We then performed analysis using spectrometric techniques. All the data as well as some graphs representing the data are given below.
Now that we had laboratory-established data for 19 UTR constructs with reporter genes RFP, we got curious about the effect of the same UTRs with other gene sequences. So, we decided to use amilCP to clear our curiosity. We decided to insert 12 of the existing UTR sequences and repeated the protocol to obtain recombinant vectors. However, to our disappointment, we figured it all had been in vain. During our analytical procedure, we realized that the fluorescence and optical density ratio was always 1. This was because the Lambda max of amilCP is 588 nm, and optical density is read at 600 nm. This leads to a significant overlap of the peaks. All our test experiments were performed in duplicates.
Test Results for DH5α Strain:
For first 8 UTRs of DH5α:
Gel Confirmation for first transformation of 6 UTR constructs in the first 8 UTRs:
For next 11 UTRs of DH5alpha:
Gel confirmation for transformation of 11 UTRs + control of the next batch:
Master plate consisting of 11 of the transformed E.Coli DH5α strain + control:
10 of our constructs + control:
10 of our constructs streaked on agar plates:
10 of our constructs + control inoculated for analysis:
