Model
Our dynamic mathematical models
and computer simulations.
Wet Lab Notebook
Here we provide a list of all the experiments conducted as a part of our wet lab and the protocols we referred to. Our protocols were compiled by conducting literature searches and other sources. We would like to express our sincere gratitude to Douglass Marr and Andrew Sillato for sharing their lab protocols and resources, and for their unconditional support throughout our project.
Complete Lab Protocols
Please click the link below to access our detailed lab notebook!
Below is a list of our protocols
A timeline of our experiments can be found below.
Timeline
Gene and Vector Amplification (July 6 - July 20)
The gene of interest PROS was amplified using PCR. We ran a gel to check if the PCR was successful. Cultures of DH5-alpha E. coli cells containing 2Bc-T plasmid were grown overnight and miniprepped. Concentrations of the gene and plasmid were recorded. The miniprepped 2Bc-T plasmid was then amplified by PCR and we ran a gel to check if the PCR was successful. This process was repeated and troubleshooted multiple times.
Restriction Digest of Vector (July 25)
The 2Bc-T plasmid was linearized using the HpaI restriction enzyme and confirmed by a gel.
LIC Reaction for Insert (July 26)
A LIC reaction was performed for our PROS insert and concentration was confirmed.
Vector Growth and Purification (July 26 - July 27)
Cultures of DH5-alpha E. coli cells containing 2Bc-T plasmid were grown overnight and miniprepped. Concentrations of the plasmid were recorded.
Restriction Digest of Vector (July 27 - August 2)
The 2Bc-T plasmid was linearized using the HpaI restriction enzyme and confirmed by a gel. This process was repeated and troubleshooted to ensure linearization took place.
Extraction of Linearized Vector (August 2 - August 3)
The linearized 2Bc-T vector was extracted from the gel using gel extraction and concentration was confirmed. A LIC reaction was performed for the gel extracted and linearized vector.
Annealing of Vector and Insert (August 4)
The linearized 2Bc-T vector and PROS gene of interest were annealed to produce a recombinant vector.
Transformation into DH5-alpha E. coli Cells (August 4 - August 14)
The recombinant vector and a positive control vector (only plasmid) were transformed into DH5-alpha E. coli cells to determine if the recombinant vector was made successfully. This was initially unsuccessful so all of the previous processes entailing making the recombinant plasmid were repeated. On the second try, there were colonies present, indicating successful transformation.
Transformed Cells’ Growth & Purification (August 15 - August 17)
The colonies from the transformation were grown overnight and miniprepped. The concentration was measured.
Digestion of Recombinant Vector (August 17)
We performed a restriction digest of the recombinant vector with Xho1 restriction enzyme. We ran a DNA gel with the product to ensure annealing took place, which yielded positive results and indication that the recombinant vector was present in the samples.
Amplification of Recombinant Vector (August 18)
A PCR of the miniprepped recombinant vector was performed. We ran a DNA gel to ensure that the samples contained the recombinant vector, which was successful.
Transformation of BL21 and Origami E. coli Cells (August 19)
The recombinant vector was transformed into BL21 and Origami E. coli cells for protein expression. The transformations were successful.
Growth of Transformed Cells (August 23 - August 24)
Cultures of Origami and BL21 E. coli cells containing the recombinant vector were grown overnight.
SDS-PAGE and Western Blot (August 27 - September 3)
Analytical techniques such as running an SDS-PAGE gel and Western Blot were performed to ensure that Protein S was made. The HRP anti-His 6 antibody successfully detected the Protein S target band at about 80 kD.
SF9 Wet Lab Notebook and Protocols
Please click the link below to access our detailed lab notebook!
Below is a list of our protocols
- PCR Protocol Optimized for the PROS Gene and Colony PCR Protocol
- Touchdown PCR
- Ligase Independent Cloning (LIC)
- Restriction Digest
- 6XHis Lyticase Purification Protocol
- Competent Cell Preparation
- E. coli Transformation Protocol
- Thermo Scientific Gel Extraction Kit Protocol
- SF9 Cells Passaging Protocol
- Primer Design Guidelines
- Monarch PCR and DNA Cleanup Kit
- SF9 Transfection Protocol
- Ethanol Precipitation for Concentration of DNA Samples
- Bac-To-Bac Baculovirus Expression Systems
A timeline of our experiments can be found below.
Timeline
PCR of new primers onto the PROS gene and the YMBacII vector (July 1-6)
We ordered the GOI with other primers attached to its ends. We need to use different primers, however. The new primers are compatible with YMBac II plasmid, into which the GOI (with new, compatible primers), will be PCR-ed into. We used Protocol for Q5® High-Fidelity 2X Master Mix. We ran a DNA gel with both products of the PCR reaction (modified YmBac II and modified PROS) to see if they have been amplified and have correct weight. They had the correct weight and good amplification, but there was significant laddering for the gene, so we had to optimize the conditions. We then performed PCR product clean up and nanodrop to determine the concentration of the gene and the vector. We made calculations to find the volume of cleaned up PCR product needed for LIC.
Making Ampicillin LB agar plates to grow the transformants on, and plating Nova Blue E. coli with the plasmid and gene cloned into them (July 7-10)
We saw no growth on any of the plates, so we decided to perform a positive control using intact YMBcII.
Running a positive control using intact YMBacII plasmid: transforming Nova Blue with intact plasmid and plating them (July 11)
Again, we saw no growth which suggested that the mistake is with either our gene and vector or with the technique of transforming or the cells.
Re-adding primers to the gene and to the vector by PCR (July 12)
We ran a gel of the results, and the bands were extremely faint, so we decided to troubleshoot the amplification and optimize the PCR.
Optimization of PCR for both vector and gene (July 13 - July 26)
We recalculated annealing temperature with corrected values, and tried different annealing conditions such as 72°C, 66°C, 62 °C, 55°C, increased annealing times, and using GC enhancers. At times our PCR reactions failed completely as proven by nanodrop, which we assumed was either human error or machine error. This could also be due to the fact that our primers were highly GC rich and could not be redesigned because of the vector we had to use. The PCR reaction was very difficult to get to work, but at last we found the perfect conditions which allowed reproducible good amplification, and minimized laddering. However, we still had to gel extract since the laddering was still significant. We managed to gel extract at very high concentration and purity (vector concentration 47.3 ng/uL and purity 1.83 where 1.80 is the ideal purity, as well as gene concentration of 56.8 ng/uL with 1.87 purity), which is more than enough for the purposes of LIC, especially that we had about 20 ul of the gel-extracted vector.
LIC (cloning the insert into the vector) and transformation of the LIC assembled vector into Nova Blue E. coli (July 26-August 3)
The first attempt was unsuccessful, so we tried it again with new conditions (lowered the time of heat shock and ice incubation). Again, no colonies formed for the second time either. For the third time, we did achieve two successful colonies. We cultured them, but the cells did not grow in culture. We tried a colony PCR to verify whether the cells had the correctly cloned insert + vector, and ran a gel. The weight of the vector that was supposed to be achieved was clearly visible on the colony PCR gel. This partially supports the claim that we achieved engineering success in creating a construct optimized for the furthering of SF9 protein S expression. To have better evidence, the next step would be to purify enough of this vector by mini-prep to be able to sequence it. Once we would sequence it, we would be sure about the success of the transformation. Further steps could include checking if we need to design primers for sequencing, and making a new 5 ml LB colony so we can sequence, since we would need to get 300-400 ng of vector to sequence. However, at this point in the experiment we did not have the time or resources to continue with SF9 and we decided to continue solely with E. coli expression.
