Results

In this page you will find details about the project's results and challenges.

Challenges

AlgD promoter

When optimizing the AlgD promoter, it was observed that our promoter's sequence did not match the one synthesized. It was also discovered that the AlgD promoter lacked any regulators, including the sigma 22 regulator, which serves as the recognition site for transcription to begin. This implies that this promoter would never have been active.

pSB1A3 and pSB1K3

While using iGEM’s linearized plasmids, pSB1A3 and pSB1K3, we were unable to obtain a ligated construct resulting in the redesigning of our cloning plan. Upon failure, we observed that the pSB1A3 plasmid did not generate the predicted EcoRI/PstI digest, and that pSB1A3 and pSB1K3 were incompatible with various RFC. We also predicted that because of the various freeze-thaw cycles it had endured since it had been acquired by the previous cycle, the DNA could have been degraded by nucleases and is therefore no longer viable for use. As a result, we modified our cloning technique for Devices 1 and 2, opting to use the pSB1C3 vector for every assembly.

Overhang compatibility

While performing the second cloning strategy for Device 1, we quickly realized that digesting the backbone (D1P1) with only one enzyme could cause multiple mistakes when ligating. Not only could the vector quickly recircularize since it has compatible overhangs, the inserts could be ligated backwards since both insert overhangs are compatible with the SpeI restriction site. On account of this realization, we changed our cloning strategy for Device 1 by digesting D1P1-BB with two enzymes, SpeI and PstI.

Inoperative T4 DNA Ligase and T4 DNA Ligase Buffer

Throughout our time in the laboratory, we were able to obtain definitive results for various protocols including miniprep, digestion reactions, agarose gel purification, bacterial transformation, among others. However, we were unable to successfully ligate any constructs. We presumed that the T4 DNA Ligase Buffer was not working efficiently due to lack of ATP, caused by multiple freeze-thaw cycles. Unfortunately, while we were ligating the A1 construct (D1P1-BB and D1P2) with a new T4 DNA Ligase and T4 DNA Ligase Buffer, we coincided with the arrival of the devastating Hurricane Fiona which left our lab without power for two weeks, leaving many materials compromised and our project without conclusive results.

Figure 1. Unsuccessful ligation of D1P1-BB [SpeI-HF] positive control (09/13/2022). In order to test if the Hi-T4 DNA Ligase we were using was working, two controls were performed. These were plated on LBA supplemented with chloramphenicol. The positive control consisted of D1P1-BB linearized with SpeI-HF then ligated with NEB’s Hi-T4 DNA Ligase. On the other hand, the negative control included D1P1-BB linearized with SpeI-HF but in the ligation reaction, Hi-T4 DNA Ligase was substituted for Nuclease free water. Absence of bacterial growth was expected on the negative control, yet not on the positive control. Lack of colony growth after 24 hours on the positive control was indicative of inefficient DNA Ligase activity.

Project Achievements

Device 1: AHL and LuxR protein Generator

Successful results

Confirmation of D1P1, D1P2 and D1P3 parts.
Successful plasmid purification, digestion, gel purification and transformation protocols.

Figure 2. D1P2 grown colonies on LBA plate supplemented with chloramphenicol (08/09/2022). EC100™ cells were electroporated with the digested D1P2 [XP] product. The plate was observed after 24 hours of incubation at 37ºC. Once colonies were inoculated in LBB and the QIAgen miniprep was completed, agarose gel electrophoresis was performed as seen in Figure 3.

Figure 3. Successful D1P2 [XbaI and PstI-HF] digestion (08/11/2022). Digestion products were separated by electrophoresis on 1% agarose and compared to NEB’s 1 kb+ DNA ladder in the first lane. Expected weights confirm digestion of D1P2, observed in the third lane. D1P2 C2 was the second colony inoculated in LBB from the plate shown in Figure 2.

Figure 4. Successful digestion of D1P1 [SpeI-HF and PstI-HF] and D1P3 [XbaI and PstI-HF] (09/08/2022). Digestion products were separated by electrophoresis on 1% agarose and compared to NEB’s 1 kb+ DNA ladder in the first lane. All lanes were consistent with their expected insert and vector weights. For the second and third lane, D1P1 [SP] was located around the 3,000 bp line since the plasmid was linearized and did not lose the insert. Lanes 3, 4 and 5, D1P3 [XP], showed weights nearing 2070 bp for the pSB1C3 vector and 999 bp for the D1P3 insert.

Unsuccessful results

Ineffective ligation of A1.

Device 2: RDX Biodegradation

Successful results

Successful plasmid purification, digestion, gel purification and transformation protocols.

Unsuccessful results

Ineffective ligation of B1.

Figure 5. Unsuccessful D2F1 + pSB1K3 ligation attempt on LBA plate supplemented with kanamycin (08/19/2022). EC100™ cells were electroporated with the ligation product. After 96 hours of incubation at 37ºC, colony growth was not observed.

Figure 6. Unsuccessful B1 ligation (D2F1 + pSB1C3) attempt on LBA plate supplemented with chloramphenicol (09/09/2022). EC100™ cells were electroporated with the B1 ligation product. The plate was observed after 24 hours of incubation at 37ºC. Upon examination, we realized that the second B1 ligation attempt was unsuccessful and a contaminant was present.

The repository used to create this website is available at gitlab.igem.org/2022/rum-uprm.