Wetlab
Cycle 1: Cloning (Engineering of E.coli BL21)
Design
Primer design of 1st and 4 sushi
The B5R protein is generally produced in eukaryotic cells in E.coli. Therefore, apart from synthesizing the full sequence, we planned to
synthesize the first sushi ectodomain of B5R. Since we need a proper
expression system for aptamer and antibody testing, pET23a was suggested
as a satisfactory plasmid vector. So, we made three primers:
Figure 1. Plasmid design of 1st sushi and 4 sushi B5R in pET23a constructs
Figure 2. Three primers for 1st sushi and 4 sushi B5R proteins
Build
PCR amplification, Preparation of competent DH5a cells, Restriction, Ligation, PCR purification, Transformation, Plasmid extraction, Colony PCR, Gel extraction
Inserts from pVAX and pET23a vector were firstly restricted and purified. Then it was transformed into BL-21 E. coli after ligation. Colony PCR and sequencing were done to select the DNAs with successful assembly.
Test
DNA sequencing on Genetic DNA Analyzer
The results of PCR colony and sequencing demonstrated proper ligation of vector and insert.
Figure 3. Three possible reasons for failed experiment
Learn
Troubleshooting from benchling
For this cycle, we learned that we have successfully assembled the construct. Moreover, after brainstorming problems from our lab, we learned that:
- We need to add a small amount of water (30ul) instead of elution buffer
- After analyzing different protocols, we decided to incubate ligation products for 20min
- The suggested volume in protocols for SYBR safe was high which made bands invisible under UV; thus, we decided to add only 5ul of it
Cycle 2: Protein expression
Design
144 different conditions for control
The preliminary data from our superuser group demonstrated that the protein expression was not observed in SDS-PAGE electrophoresis. Thus, we decided to check the expression of B5R protein in BL21(DE3) E. coli under different conditions. The conditions varied depending on time (2h,4h,6h,16h); temperature (19°C, 37°C, 37°C); IPTG concentration (0mM, 0.2mM, 0.5mM, 1mM).
Build
Samples were prepared for test expression with SDS-PAGE
We run SDS-PAGE and Western Blot electrophoresis in BioRad protein electrophoresis.
Test
Protein B5R was not expressed
To test electrophoresis gels, we searched for significantly large bands around 6 and 25 kDa for 1st and 4 sushi B5R proteins, respectively. Also, the bands were analysed in Western blot for proper protein folding.
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Reasons why:
We did not find any large observable bands in gels. The present small bands around the 6 and 25 kDa area were not supported by Western blot. This means that there was no or minimal expression of our protein. The possible reasons can be:
- Toxicity of B5R protein to BL-21 E. coli which kills them without allowing for observable protein expression. Thus, we suggested inserting the signal sequence for Sec (secretion) system into our B5R sequence.
- The failed expression system in our pET23a plasmid. It can be possible that our sample of initial vectors may have a defective T7 expression system.
- Another issue may result from non-optimized B5R codon sequences for BL21 E. coli.
Thus, we suggested using a different plasmid pET22b plasmid with an already inserted signal sequence. This way we can tackle the two above-mentioned problems. Also, we planned to order codon-optimized B5R sequences. However, we did not have enough time to finish these new suggestions until the deadline for the wiki freeze. Therefore, we focused mainly on three other antigens (L1, A27, A33) to test our optic fiber biosensors. Since aptamers can be used instead of antibodies, we planned to functionalize them on antibodies as well.
Drylab
Cycle 3.1: Optic fibre functionalization with L1, A27, and A33
Design
Designing optic fibers
Optical fibres are an innovative way to make biosensors to detect specific molecules in different media. They are cheap, sustainable, extremely safe and experiments have shown high sensitivity of detection - up to attomolar 10-18 concentrations of molecules. Ordinary optic fibres can be turned into biosensors by attaching antibodies of the target molecules onto them in a process called ‘functionalization’, then immersed into solutions with varying concentrations of the target proteins, and their responses can be analysed
Build
Functionalizing optical fibres with antibodies of proteins of Vaccinia virus
Since protein B5R was not expressed in our engineered E.coli, the optical fibres were functionalized with antibodies of other proteins of Vaccinia virus: L1, A27 and A33.
Test
Detecting Vaccinia virus proteins using optical fibre biosensors
We conducted tests with two types of optic fibre biosensors: biosensors functionalized with single antibodies and multiple antibodies (multiplex biosensors). Biosensors with single antibodies have been tested for detection of their antigen in PBS solution, while the multiplex sensors were tested both in PBS and sewage water. The results were analysed using the software our team has devised (see here).
Figure 4. Test of biosensors with single and multiple antibodies for antigen detection in PBS and sewage water.
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Optical fiber biosensors detect proteins and changes in their concentrations in PBS and sewage water
Overall, the optical fibre ball resonators functionalized with single antibodies and the multiplex biosensors succeeded in detecting the changes of concentrations of the target antigens in PBS with high sensitivity. Furthermore, the multiplex sensors were observed to successfully detect antigen’s concentration changes in samples of synthetic sewage water, indicating that it was functional at conditions close to the real environment.
Cycle 3.2: Aptamer designing
Design
Using aptamers instead of antibodies
Aptamers are nucleotide sequences that specifically bind to target molecules, and have advantages of being cheap and accessible over antibodies, and thus it is reasonable to functionalize optical fibres with aptamers and test them on detecting the target molecules. The conventional way to make aptamers is via a laborious process called SELEX that usually takes a long time, and there is open-source software MAWS made by iGEM Heidelberg 2015 team that we can use to make aptamer sequences for vaccinia virus proteins.
Build
Using MAWS to make aptamer sequences
We used the software MAWS to make DNA aptamer sequences for a Vaccinia virus protein L1 (as we had limited time, for one protein only).
Test
Conducting in-silico tests on aptamers
Then we made the 3D structures of the DNA aptamers and simulated a docking model with aptamers and their target molecules and analysed possible interactions they might have.
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Aptamers are ordered for in-vivo tests
The results show low binding energy, high confidence scores, high rmsd values and seemingly good amount of hydrogen bonds in the binding of aptamers and the protein. However, the results are rather ambiguous, and it is hard to say if the aptamers and proteins form a good complex with high confidence, therefore, for reliable validation of the aptamers, we ordered them to conduct further in-vivo tests on their binding.