Team:Virginia/Engineering
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Engineering
Index:
Engineering Design Cycle
Design of the Manifold System
Designing the Manifold system went through numerous different phases during the initial planning of the system's parts. From modeling to design to experimentation and verification, the design of each part was verified, judged, and constructed. We can’t pretend that our careful planning made this process go off without a hitch; in fact, the trial and error of the engineering design cycle was a necessary part of improving our project.
Trial and Error
The best example of our engineering design cycle is our reverse transcriptase (RT) part. RT is necessary to transcribe the RNA of our scaffold genes back into DNA, which can then bind to our zinc finger-fusion enzymes. We originally planned to assemble our RT part from standard plasmids that could be ordered from places like Addgene, thus saving money on gene synthesis. Once these standard plasmids were obtained, we originally hoped to assemble the entire system through a series of PCR reactions and ligations.
When designing our DNA scaffolds, we learned from the paper Genetic Encoding of DNA Nanostructures and their Self Assembly in Living Bacteria
[1]Elbaz, J., Yin, P. & Voigt, C. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun 7, 11179 (2016). https://doi.org/10.1038/ncomms11179
that a combination of HIV-RT and MLRT produces the highest yields of DNA scaffolds, compared to just using one RT or the other. This required combining the two RT parts into a single plasmid.
HIV-RT and MLRT were available separately from Addgene; however, each plasmid contained illegal restriction sites. We planned to use mutagenesis techniques to remove these illegal sites, then an overhang PCR reaction of the RT genes, before finally ligating the RT parts together with a promoter, Lac(O), and terminator.
Unfortunately, our initial assembly plans were not as successful as we had hoped. Part plasmids were correctly amplified through PCR, but combining these plasmids into a single cohesive part was not successful. Several attempts were made to combine these parts, however verification via culture, DNA extraction, restriction enzyme digest, and agarose gel electrophoresis of these attempts showed that we were still unsuccessful.
Reappraoching the Design
Seeing that our first approach was failing to produce the desired results, we decided to change tactics. We realized that given the critical importance of our RT gene and the amount of time we were spending on creating it, we would be justified in spending the money to get it sequenced de novo. We decided upon Golden Gate assembly as the best way to put the two HIV-RT subunits and the ML-RT gene together, since we could accomplish it with a single enzyme and in a single step. We performed the assemblies once the parts arrived, ligating them into a linearized pSB1K3 plasmid with standard BioBrick restriction sites.
Verifiying the New Approach
In order to verify these assemblies, we transformed E. coli cells, cultured them, and extracted their DNA with a miniprep. We then conducted a restriction enzyme digest using BioBrick enzymes and analyzed the resulting fragments on an agarose gel. The bands appeared in exactly the locations we had predicted, indicating that our second attempt was a success.
Fig 8. Left: The RT Full Part digested with XbaI and SpeI (expected sizes 2196bp and 5645bp). Lanes 5, 6, and 7 each have a different sample of the same RT Full Part plasmid. Right:The RT full part digested with only Xbal (expected size 7841bp). Lanes 5, 6, and 7 correspond to different samples of the RT Full Part plasmid, and are taken from the same samples, respectively, as lanes 5, 6, and 7 in Figure 2.3.
Success is not the End
From these trials and errors, we were successfully able to completely assemble the Manifold system. But our modified reverse transcriptase is only one part of the full Manifold system, which we are currently working to assemble and test.
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485 McCormick Rd
CHARLOTTESVILLE, VA 22903
485 McCormick Rd
CHARLOTTESVILLE, VA 22903
