Adding to existing Parts by Literature research
Since our project aims to detect and decontaminate manganese pollution, we wanted to create a bifunctional system that would manage both tasks simultaneously. We picked two parts brought to the registry by Team Calgary in 2012: The manganese riboswitch (BBa_K902074) and a manganese-inducible promoter (BBa_K902073). For our own project design, and to contribute to the parts documentation, we deepened our understanding of these parts through literature research.
Dambacher et al. published a paper in 2015 revealing more details of E. coli manganese regulation. In summary, the regulation of the MntP manganese exporter consists of two regulatory elements. The first element, a promoter, enables transcription of MntP when activated by the regulatory proteins mntR and Fur, which in turn only bind/activate the promoter in the presence of relatively high intracellular manganese concentrations. The second element, a riboswitch located in the 5’ untranslated region (UTR) of the MntP mRNA, undergoes structural changes upon binding manganese. The structural change increases the ribosomal binding site accessibility and allows for translation initiation . These and more findings helped us design our experiments and also inspired new ideas for future experiments (see Experiments and Outlook). Therefore, these findings were added to the corresponding part pages.
Adding new Basic and Composite Parts
We created a new basic part, the FAST2-tag (BBa_K3510000), and combined it with several already existing parts in the registry, which resulted in four new composite parts for our PacMn project (BBa_K3510002-5). For our side project, the Vitamin B12 biosensor, we designed two new composite parts (BBa_K3510006/-7). For more information, visit our wiki pages Design and Parts.
After iterating through the Build - Test - Learn - Improve cycle, we successfully cloned all four of our PacMn project composites in E. coli and reached the next phase: Testing their functionality. We quickly noticed that our clones were not performing as expected. A reexamination of our sequences revealed a mistake in our design that hindered protein expression: A missing ribosome binding site between the riboswitch and our gene of interest. Unfortunately, due to this year’s minimal time and space available for wet lab work in comparison to previous years, by the time we realized what the problem was, it was already too late to fix the sequence. Nevertheless, we rapidly came up with a plan B to test the inducibility of our manganese promoter (see Results). Although the result for this experiment was inconclusive, and our time had run out, we created one last opportunity by joining our efforts and skills.
Our project, although not completed as planned, was successful in many other ways. Our frequent encounter with problems promoted our creativity and gave birth to many ideas that were either immediately implemented or formed part of our Outlook. Some of these ideas were also added to the registry entries. Hopefully, future iGEM teams use this knowledge as a foundation or as inspiration for new projects.
Troubleshooting in the Lab
While executing our wet lab work, we ran into multiple problems with cloning-related experiments. Since we were able to overcome these problems by applying the Design-Build-Test-Cycle, we want to share what we learned from this experience so that future iGEM teams have a starting point when approaching troubleshooting. This short document describes the troubleshooting techniques that we implemented in our experiments and should only be taken as suggestions rather than as rules.
Agarose Gel Electrophoresis
- Faint bands or in need of time: Switching to precast DNA staining gave us clearer results and saved us time that was lost in the 20 min incubation time required for stain baths.
Polymerase Chain Reaction (PCR)
- Intense primer clouds: Reduce primer concentration to 25%-50%.
- Unspecific gel bands: Gradient PCR (higher temperatures) or gel extraction and purification of the specific band.
- No specific band: Switch to a more robust PCR kit/polymerase, but expect higher mutation frequencies (lower fidelity) (e.g. from UCP HiFidelity polymerase to AllTaq polymerase).
- More yield: Increase template DNA concentration, primer concentrations, or cycles. Be careful, as these modifications may increase unspecific amplification or mutation rate.
- Unsure of how many colonies to expect: Perform a parallel Gibson Assembly experiment exclusively with linearized/digested backbone DNA. This will give you an idea of empty vector background due to unlikely re-ligation events or improper backbone digestion.
- Too many colonies after transformation: Plate 100 µL of the reaction.
- Low assembly efficiency: Use less of the reaction product by diluting it 1:5. Add smaller fragments in a higher insert-to-backbone molar ratio than larger fragments (5:1 vs. 3:1).
Economic approach to RT-PCR
- No quantitative PCR instrument: You can roughly quantify the template cDNA by pausing the PCR every certain number of cycles to extract a small part of the PCR reaction and load it on an agarose gel. In the end, run gel electrophoresis and compare signal strengths at each interval.
- No lysozyme to lyse the cells: The Qiagen buffers P1 and P2 (or equivalents) for plasmid DNA isolation can also be used for lysis of Gram-negative bacteria for RNA isolation. However, P1 can only be used if RNase has not been added.
- Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.