Team:Chalmers-Gothenburg/Contribution
Characterization of BioBricks
The Improved Biosensor Project
When designing our project, we took inspiration from the iGEM team from Paris-Saclay 2013. This team managed to create a biosensor in Escherichia coli which was able to detect PCB, and we realized that this BioBrick could be a great addition to our project as well. By combining this with our intended BioBricks we could create a system that could sense, report and degrade PCBs.
With this project we aimed to improve the previous biosensor by changing the reporter. The previous reporter system included the LacZ gene encoding the β-galactosidase enzyme, which was then monitored by a chemical reaction using Xgal from which blue light was emitted. The β-galactosidase was expressed under the promoter bphR1, which was activated when a protein called BphR2 first bound to PCBs and then to the promoter as a transcription factor. The gene bphR2 was located upstream of the reporter and expressed under a constitutive promoter, which meant that the β-galactosidase activity correlated with the presence of PCBs. Our intention was to replace the reporter gene with a Green Fluorescent Protein with a degradation tag instead. This would yield a more dynamic system able to respond to changing levels of PCBs, as when PCBs would be degraded the GFP would also be degraded, leading to decreased fluorescence. An illustration of how the biosensor would be constructed is shown in Figure 1.
To do this we used a GFP BioBrick, a promoter and a terminator that were present within the iGEM 2019 DNA distribution kit. These are displayed in Table 1.
| Plate | Well | Name | Description |
|---|---|---|---|
| #1 | 3O | BBa_K608002 | Promoter |
| #1 | 1B | BBa_K731722 | Terminator |
| #5 | 8G | BBa_K1399005 | Green fluorescent protein (GFP) |
The DNA sequences for the BioBricks made by the Paris-Saclay 2013 team mentioned above were found on the iGEM parts registry website (BBa_K1155009 (bphR2), BBa_K1155001 (bphR1)), and the sequences were ordered from Twist Bioscience.
The intention was to construct the new composite system through Gibson Assembly. Therefore, the BioBricks were amplified using primers designed to create overhangs between the different parts. To view all results in detail, please refer to the lab notes for the biosensor project found on the notebook page. Unfortunately, the BioBricks provided by Twist Bioscience could not be amplified even after multiple attempts. We suspected that something might be wrong with the template, since none of the PCR attempts produced any results. Our team worked hard in the lab trying different settings for the PCR, changing the primer working stocks, making sure that all fragments were the correct ones and consulting with supervisors. As a troubleshooting measure, and the lengths of the ordered fragments were verified through restriction digestion, and both of the fragments showed the correct lengths. The reason that the fragments could not be amplified could be errors in the primers or in the sequence given. As the time of iGEM is short, there were no time for our team to order new fragments form Twist Bioscience and so the improvement of the BioBrick could not be attempted.
Characterization of a Luciferase BioBrick
As the previous characterization attempt had failed, we had to come up with a new idea. Within the iGEM parts registry we found a BioBrick encoding a luciferase enzyme, created by the iGEM team of Tokyo-NoKoGen 2012. This team had created a BioBrick consisting of a LuxCDABFEG and a filter.
The part used was sent to us by iGEM HQ in the 2019 distribution kit, and the details about it are displayed in Table 2.
| Plate | Well | Name | Description |
|---|---|---|---|
| #5 | 13E | BBa_K769020 | Luciferase |
The evaluation of the BioBrick was done according to the wiki of the iGEM team Tokyo-NoKoGen 2012. On their wiki it is specified that adding substrate is not necessary and that light emission occurs automatically.
To view all results in detail, please refer to the lab notes for the luciferase project found on the notebook page. The BioBrick was successfully transformed into E. coli TOP10, the same strain that was used by the original team. The same procedure was then carried out as specified on the Tokyo-NoKoGen wiki. Transformed E. coli TOP10 was precultured and L-arabinose (0.2% f.c.) was added to induce and express the proteins. The culture was incubated at 20 ℃ as iGEM Tokyo-NoKoGen suggested on their wiki.
Multiple attempts were carried out but without any sign of bioluminescence. This result was unexpected since the transformation was verified using negative control, the procedure was according to the iGEM Tokyo-NoKoGen wiki description and the culture was incubated at optimal temperature. Unfortunately, this BioBrick does not seem to work.
The BioBrick was sent for sequencing. The results revealed a missing base pair in the promoter region, however it is unlikely that this affects the expression if it is not in the polymerase binding region.
The sequencing results also showed a potentially lacking base pair in the operon, see Figure 3. The credibility of these reads is questionable, since it is at the end and beginning of each sequencing primer’s range; it is also part of a poly-A sequence. However, since the gap is present in both reads we still thought it would be worth mentioning. If a base pair is indeed missing, this would cause a frameshift, making a large part of the operon useless.
Other Contributions
The improved biosensor, as we envisioned it, could in fact have different kinds of reporter genes. For this purpose, we tested several different reporter genes provided in the iGEM DNA distribution kit, including green fluorescent protein (GFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP) and blue chromoprotein. These BioBricks are listed in Table 3, along with the high constitutive expression casette used to test them. In addition, the promoter and terminator displayed in Table 1 were also used during the testing.
| Plate | Well | Name | Description |
|---|---|---|---|
| #1 | 1G | BBa_K314100 | High constitutive expression casette |
| #4 | 9I | BBa_K156009 | Orange fluorescent protein (OFP) |
| #5 | 10B | BBa_K1692032 | Blue chromoprotein with RBS and promoter |
| #6 | 17J | BBa_K1418040 | Cyan fluorescent protein |
The BioBricks provided by iGEM HQ were amplified and tested. To the BioBricks lacking promoter and/or terminator we added these parts using the iGEM restriction and ligation method. The BioBricks were ligated by 3A-assembly, which failed for OFP and CFP. For GFP, while there were positive colonies on the selection plate, the colonies did not show fluorescence under a fluorescence microscope. Further trials and verification of these BioBricks was not possible, due to unavailability of enough pSB1A3 to continue experiments; hence, we could not express the BioBricks. For details about results from these experiments please refer to our lab notes.
Further Reading
Check out the rest of our lab-related work by clicking on any of the images below! Go over the protocols used for the different types of Experiments performed, scroll through all of the 4 months of lab work documented in Notebook, read up on the details of the different Parts used, or go straight for the actual Results of all our hard work. Safety will take you to the details of how we made sure to keep ourselves and others safe in the lab.
