The Engineering Cycle as a Fluid Model
We started out with a design stage in wetlab engineering cycle, where we proposed a plasmid. At the time we weren't sure we would be able to gain access to a GMO lab, so we jumped into a drylab engineering cycle to produce a software tool that could help us steamline the docking process. With this tool we found problems with our plasmid and therefore started a new wet lab engineering cycle. This is just one of the examples of how wetlab and drylab have helped each other in our project.
Figure 1: The engineering cycle used with our project.
The Ⅰ Engineering Cycle in Wetlab
In the first phase of the IGEM Wetlab engineering cycle, the design stage. We attempted to design a plasmid. We made a general plasmid with the general structures. It was important for us that the RBS had strong affinity to produce many proteins, and that the protein contained a means for protein purification, so that if we ran into problems and wanted to examine the protein, that was a possibility. Having a selection marker was also important as it meant having an automatically integrated test for whether the bacteria had been transformed or not.
Figure 2: Our plasmid in the first phase of the engineering cycle.
From there we researched specific parts and built our vector with a vector builder. We found that the pBAD promoter met almost all of our requirements for a plasmid and is generally known as well used and reliable plasmid. The only thing missing was our gene of interest as well as a means for protein purification.
Hence we added our gene of interest, the gene for oxalate oxidase. Upstream of this gene a HIS-tag as well as TEV was added. In this way we could do protein purification with the HIS-tag if we ran into problems and wanted to examine the protein isolated, as well as being able to remove the his-tag with TEV proteases that can recognize the TEV sequence and cut.
Figure 3: Our plasmid in the second phase of the engineering cyccle.
To avoid buying all the parts building and testing, and then realizing something was off. We decided to test our gene products with bioinformatic tools. This can be a rather tedious process and with this in mind we started a new engineering cycle, an in silico engineering cycle where we would attempt to simulate the chances of the enzyme encountering natural inhibitors within the body.
The in Silico Engineering Cycle
In drylab, we used the engineering cycle as a fundamental model to develop the tools that are called LightningAutoDock Tools - in short LAD tools. This was done using this engineering cycle model by first designing and generating the ideas and the functionality that we wanted to implement into our final design to complete the docking tasks. After testing the LightningAutoDock (the python tool running the docking software) and seeing errors such as cmd prompt freezing, instability and slow docking speed, we corrected the script in order to address the issues.
Along with this iterative process there were a lot of debugging, a lot of miscommunication between the AutoDock4 algorithm files and the way it parsed the arguments. This meant that we used the engineering cycle in drylab by first designing, then building, then testing and then learning. After many iterations we had acquired sufficient knowledge of how the AutoDock4 systems worked and then the development of the python tools were easier to complete. This process was done over many weeks in order to finalise the LightningAutoDock Tool suite.
During our docking simulations, we made a significant discovery regarding histidine's ability to bind at the entrance of the active site of our enzyme, oxalate oxidase. The conformation with histidine and the oxalate oxidase had a binding energy of -7.4 kcal/mol. This posed a potential issue, as our purification strategy initially relied on a 6xHis-tag (six histidine residues in sequence). Given the theoretical risk of histidine residues from the tag interfering with enzyme activity, either by blocking the active site or causing aggregation, we decided to replace the His-tag with a GST (Glutathione S-transferase) tag. This switch allowed us to avoid possible complications with enzyme function during purification, even though we were unable to fully implement the GST tag experimentally within our project’s timeframe.
The II Engineering Cycle in Wetlab
Design
With our learned knowledge from our in sillico engineering cycle we once again attempted to make a wet lab engineering cycle and designed a new plasmid. In the in sillico engineering cycle we produced a new software tool and used it to learn that we could not have a HIS-tag on our final product was not viable. So we replaced the HIS-tag with the GST tag, tested the redesigned gene product with the in silico software tool and found no pressing problems. With this in mind we started the building stage.
Figure 4: Our final plasmid design
Build
We had trouble finding a GMO lab to house our experiments. Therefore, we ordered a pre assembled plasmid form vector builder. For the transformation of the bacteria we used heat shock. and set them to grow our agar plates. With that we had build our “biological machine”.
Test
In the testing stage we had set up 2 tests. One general test where we tested that the bacteria had been properly transformed through an antibiotic selection marker.
For the second part of the test stage the bacteria was then transferred into a liquid medium, after having grown in the liquid medium the cells were lysed and centrifuged. In this way we got our enzyme out of the cells and the supernatant could then be transferred to a solution with oxalate to test the effects. We then through titration measured how much we were able to break down.
Learn
Our tests showed that we were able to successfully transform our E.Coli. and that the enzymes produced were able to successfully break down oxalate. More information is available on our Results page. In this way we learned that when we transformed E.Coli with this specific plasmid. The E.Coli were able to grow and produce enzymes. When these enzymes were transferred and tested on oxalate, they were able to break down the oxalate. This knowledge gives us something to build upon if we were to go into a new engineering cycle.
Hypothetically if we were to make a new engineering cycle. We might try to redesign our plasmid so it becomes fit for a lactic acid bacteria, and insert it into a lactic acid bacteria capable of yogurt. otherwise we might try to redesign the regulatory part of our plasmid in such a way that oxalate oxidase is expressed only when oxalate is present. In engineering many redesigns and possibilities for optimization lie right in front of us. and it is our choice which ones to choose.
