February 2nd to February 8th
Everyone participated in a lab safety tour with Dr. Archer. We discussed transformation, including the lab techniques involved in each step. We also laid out expectations for the project timeline and member assignments.
February 9th to February 15th
We began lab training with running 5 uL of practice PCR reactions and worked on a sequence planning activity in preparation for sequence planning required for the project. We also cleaned up the lab space and organized materials.
February 16th to February 22nd
We practiced gel electrophoresis preparation and loading. We also participated in gel analysis exercise and troubleshooting for possible errors, and reviewed gel electrophoresis applications in connection with the project.
February 22rd to February 29th
We worked on training for bacterial transformation. We also went over all the aspects of sterile technique and plating. We reviewed the steps of Golden Gate Assembly and practiced using the nanodrop.
March 1st to March 7th
We ran a Golden Gate Assembly reaction to practice the protocol. We did training for experimental design and discussed the roles that controls play in various studies, as well as how we would incorporate them into future experiments for our future project.
March 8th to April 11th
Sent home for covid
April 12th to April 18th
We did virtual transformation training by going through the steps and information we need for understanding the protocol. To practice making sequences, we searched for suitable promoters for a part of our genetic circuit. We ended training by walking through the steps of primer creation for Golden Gate Assembly using Benchling.
April 19th to April 25th
We continued our work on making sequences for parts of the genetic circuit including the toxin, antitoxin, therapeutic, and mCardinal.
April 26th to May 2nd
We finalized the lactate-inducible promoter and antitoxin sequences since we were unable to finish these sequences in the previous week.
May 3rd to May 9th
We did MATLAB training by going through demonstrations of manipulating arrays and entering equations for our models. We learned how to important data on MATLAB and use this data to generate a script. We began briefly taking a look at the differential equations we would use to model our toxin-antitoxin system.
May 10th to June 13th
Break for final exams.
June 14th to June 20th
We reviewed background information about Michaelis-Menten kinetics. We also discussed using MATLAB to model gene expression with an activator.
June 21st to June 27th
We researched the overall mechanism and lethal dose to kill bacteria for GhoS/GhoT Toxin-Antitoxin system. We also determined differential equations, using Michaelis Menten Kinetics for modeling production and degradation of toxin and antitoxin, as well as mRNA production and degradation. Finally, we found some parameters and modeled the differential equations for each component of the system using MATLAB.
June 28th to July 4th
We chose trichosanthin as our final therapeutic and modeled its basal expression, degradation and export to determine the lethal concentration required to inactivate ribosomes in tumor cells. The steady state in the bacterial cells was 35.9 µM and 0.00955 µM once it diffuses into the cancer cell. Additionally, we modelled the production of mCardinal production in E. Coli cells. We determined the steady state concentration of the mCardinal to be 0.0158 μM, using parameters such as DNA transcription rate, mRNA translation rate, and degradation rate.
July 5th to July 11th
We worked on improving our modeling for the GhoT/GhoS toxin-antitoxin system and searched for more parameters. We also researched typical metastasis locations and determined how that would impact the modeling.
July 12th to July 18th
We modeled bacterial growth in an average sized tumor and a smaller tumor (metastasis). We determined that bacteria will grow within tumor bounds with high local therapeutic concentrations for the average tumor, and will persist long term in the smaller tumors. This allowed us to show that the treatment may function in the long term until the tumor shrinks and eventually disappears. We also collaborated with Product Development to do some general information about cancer treatments.
July 19th to July 25th
We did general background research on drug delivery methods and use of other cancer treatments along with ours. We found injection to be the most efficient method, and determined that our therapeutic would work best as a supplement to other cancer treatment methods. We looked into how we could incorporate the Asd obligate gene into our plasmid and found that we could use Lambda Red recombineering, Golden Gate assembly, and subsequent plate testing. We found plasmids for this process that we would be able to purchase online.
July 26th to August 1st
We found a better toxin-antitoxin system to use for our genetic circuit: the holin-antiholin system. Our modeling showed that it would be more successful for our goals compared to the GhoS/GhoT system. We also created a diagram of our plasmid in SnapGene.
August 2nd to August 8th
We were able to find a protocol for incorporating the Asd obligate gene into our plasmid, which we may use if we are able to get into the lab. We also explored the possibility of using the LuxR to increase the expression of mCardinal to improve our fluorescence imaging abilities.
August 9th to August 15th
We worked with our Product Development team to research more information about light absorption for different skin tones. We came up with a plan of 5 plasmids that we would theoretically use in experiments, and we wrote up experimental procedures using each plasmid. The experiments we wrote up tested our improved part, trichosanthin production, and bacterial survival at different lactate concentrations.
August 16th to August 22nd
We began compiling our results from our summer modeling to put onto the Wiki. We coordinated times to enter the lab under restricted COVID-19 regulations.
August 23rd to September 12th
Break
September 13th to September 19th
Some members began going into the lab to finish the training that got cut short due to being sent home for COVID.
September 20th to September 26th
We consolidated our figures and cleaned up our MATLAB scripts.
September 27th to October 3rd
We wrote up registry pages for all of the parts that required them, including our improved kill switch, mCardinal, trichosanthin, and Asd.
October 4th to October 10th
We worked on editing all of the sections that will be included on the Wiki.
October 11th to October 17th
We finalized our drafts for the Wiki pages.