The biological system of E. coli was engineered and re-wired to produce our target enzyme. This engineering involved designing changes to its genetic make-up through the introduction of a constructed plasmid that codes information on the chosen transaminases. By using the engineering cycle we were able to define our expected outcomes and build constraints. We used the phases of the cycle to reach as close as possible to our ideal outcome while also reflecting on ways to improve our experimental design. The end of each engineering cycle prompted the start of another cycle or provided suggestions for improvements to be implemented in the future.
Cycle 1: Cloning in pET-15b
Design
To express our transaminase proteins, we needed a host vector backbone that was well characterized and capable of producing high copy numbers. Based on availability of vectors in our host lab we chose to clone our proteins into the pET-15b vector. The vector allowed for IPTG induced expression which was an effective way to control expression of the enzymes. The easy access to and the adaptability of the vector to serve our purpose of protein expression made it suitable to host our transaminase sequence.
Build
In accordance with our initial plasmid design, we planned to clone three transaminases (AATA, HHTA, and CVTA) into the pET-15b vector. This was accomplished through restriction enzyme digestion and Gibson assembly. We then transformed our plasmid into DH5-alpha and BL21 competent cells and confirmed successful cloning by full plasmid sequencing.
Test
After successfully cloning into pET-15b, we started test expressions, varying IPTG induction concentrations, and incubation temperatures and durations in order to determine the best conditions for expression. We performed pull-down assays using Ni-NTA resin, followed by SDS-PAGE gel electrophoresis to see if our transaminases were being expressed. However, initial results were inconsistent across different gels, so we performed a western blot to more conclusively determine whether or not protein was being expressed. This indicated a complete absence of any protein expression.
Learn
We believe that this was due to an indel in the lacI gene in our pET-15b vector, which was revealed by the results of plasmid sequencing. The lacI gene codes for the lac repressor protein which regulates the expression of our transaminases. The indel in the lac repressor was likely responsible for the lack of expression with and without IPTG.
Realizing that modifying protein expression conditions wouldn't rectify this vector-related problem, we decided to reclone our constructs into a different vector. We chose pQE81XN, the vector that hosted our positive control, for the recloning because, as seen in Figure 1, our positive control was expressing well, and so we were confident that recloning into this vector could lead to protein expression.
Cycle 2: Test Expressions
Design
Based on what we had learned from cycle one, we redesigned our plasmids, planning to clone into the pQE81XN vector that we knew was capable of protein expression due to how well our positive control expressed in test expressions.
Build
We cloned our transaminase inserts into pQE81XN and then transformed our plasmids into DH5-alpha and BL21 competent cells, using our same protocols from cycle 1 but integrating what we had learned from this previous iteration of the cloning cycle.
Test
After recloning into the pQE81XN vector, we again perform test expressions in order to optimize protein expression. We induced cells with 1mM IPTG at 16C for 4 hours and 16 hours. Initial test expressions (Figure 3) revealed that AATA expressed well and HHTA expressed sparingly, while CVTA did not express at all.
Learn
Our new pQE81XN plasmids expressed AATA and HHTA, but CVTA still did not express. While we tried to understand why the CVTA expression failed, we were able to optimize expression of AATA and HHTA. Test expressions showed that lower temperatures and longer incubations produced better expression than higher temperatures and shorter incubations. This permitted us to move forward with larger-scale expressions of these two transaminases.
Cycle 3: Preparing UV-Vis Assay
Design
To characterize the enzymatic activity of our transaminases, we decided to take advantage of the unique absorption spectra of the intermediates formed throughout the reaction. To ensure that the only absorption data we were collecting was a result of the transamination reaction and not the substrate's inherent absorption, we aimed to normalize the data with the absorption of individual substrates at the same concentration.
Build
To conduct the following, we used a UV-Vis spectrophotometer and prepared 500 uL reactions containing a fixed amount of enzyme and substrate.We visualized the formation of intermediates by taking absorption readings after the addition of every substrate and by tracking the same through time. To prevent unbound PLP from affecting results, we desalted the enzymes to remove any unbound PLP.
Test
The enzyme without any substrates is expected to form a peak at 411 nm as a result of the covalent binding of a protonated PLP to the enzyme’s active site as a coenzyme. However, when we first conducted the assay, we observed no visible peaks at 411 nm but instead a peak at ~ 350 nm as seen in Figure 4A. This corresponds to deprotonated PLP and could lead to an inactive active site.
Learn
To troubleshoot this problem, we decided to check the absorbance of our stock solution itself. The stock solution used for this preliminary test was from a 100mM PLP in HEPES stock which as seen in Fig 4C showed unreliable absorbance spectrum causing us to doubt the integrity of the stock solution. We also had a secondary stock of PLP in the TBS buffer which we tested on UV-Vis and observed expected absorbance (Fig 4B). We were able to infer that PLP degrades over time when stored in HEPES but is relatively more stable in TBS kept at 4˚C in the dark. Using this learning, we were able to conduct a new assay by incubating the enzymes with PLP from the stock in the TBS buffer. This cycle was a success as seen in Figure 5 since we were able to show the correct covalent binding of the enzyme to PLP.
