MatLab Modeling
From one article (Michelle E Wowk; Joseph A Trapani (2004). Cytotoxic activity of the lymphocyte toxin granzyme B. , 6(8), 752–758. doi:10.1016/j.micinf.2004.03.008), we learned that GrB starts in an inactive zymogen form which is then cleaved by Cathepsin C to be activated. It is unclear if this is happening in our model. Some questions arise if this cleavage is occuring. How is GrB getting cleaved? Is there also a source of Cathepsin C? What is the mechanism of this cleaving? Is there some complexation which then results in an active GrB? Do proteases even break down? Or are they inhibited? It seems like proteases have very long half lives. Hence, a significant assumption of our model that is being made is that the GrB produced by our circuit is already in its active cleaved state.
Table I: Parameters used in the MatLab model of the expression cassette
| Parameters | ||
|---|---|---|
| Name | Value | Source |
| Promoter strength (full): tps_active | 1/22 transcription/second | Lionnet T, Singer RH. Transcription, one allele at a time. Genome Biol. 2010;11(8):129. doi: 10.1186/gb-2010-11-8-129. Epub 2010 Aug 27. PMID: 20796323; PMCID: PMC2945777. |
| mRNA half life (for mRNA transcribed from CMVp) | 42 min | Lionnet T, Singer RH. Transcription, one allele at a time. Genome Biol. 2010;11(8):129. doi: 10.1186/gb-2010-11-8-129. Epub 2010 Aug 27. PMID: 20796323; PMCID: PMC2945777. |
| Protein half life | TNFalpha: 4.6 min GrB: Unknown | Simó R, Barbosa-Desongles A, Lecube A, Hernandez C, Selva DM. Potential role of tumor necrosis factor-α in downregulating sex hormone-binding globulin. Diabetes. 2012 Feb;61(2):372-82. doi: 10.2337/db11-0727. Epub 2011 Dec 30. PMID: 22210320; PMCID: PMC3266423. |
| Translation efficiency: eff | Unknown | |
Table II: Constants used in the MatLab model of the expression cassette
| Name | Equation | Value (rounded) |
| Average mRNA life time | mRNA1/2 /ln(2) | 60.59 min |
| kd_mRNA | ln(2)/mRNA1/2 | ln(2)/mRNA1/2 |
| kd_Protein | ln(2)/protein1/2 | 0.151 1/min → TnfA ; Unknown → GrB |
| K_transcription (ktr) | tps_active *60 | 2.73 min |
| K_translation (ktl) | eff/avg mRNA life time | N/A |
Equations taken from here.
Relevant equations:
| Name | Reaction | Reaction Rate | Notes |
| Transcription | null → mRNA | ktr | CMV promoter is constitutively activated. Assume the same transcription rate for both TnFA and GrB. |
| Translation | mRNA → mRNA + Protein | ktl*mRNA | |
| mRNA degradation | mRNA → null | kd_mRNA*mRNA | |
| Protein Degradation | Protein → null | kd_prot*protein |
Matlab Simbiology was used to model the AAVP transgene cassette. First, we can discuss the parameters. Normally they are found through experimentation in a wetlab, however the goal is to determine if the outputs of the system are sufficient to accomplish our goal; serving as a proof of concept. So instead, we have performed extensive literature searches to find accurate values. Our model simulates the path from DNA to RNA to protein. Equations, found from a previously created biomodel were used to simulate transcription, translation, and degradation of mRNA and proteins. A big that we made is that the rate of RNA translation for both TNFalpha and Granzyme B are the same. This assumption is based on the fact that we have a single CMV promoter with its own transcription rate. Overall, this provides a good overview of the expected behavior of our circuit and how it enables us to accomplish our goal.
A large assumption that we made is that the rate of RNA translation for both TNFalpha and Granzyme B are the same.
Benchling Modeling
Benchling was used extensively for in silico modeling and experimentation. Its ability to visually represent the phages and sequences was essential for us to understand their function and in designing them.
Visualization of f3-55nm-MCS. It shows crucial information for usage of the phage. The digestion sites for insertions are annotated, as well as the ligation sites and sequences from f3-55nm and fMCS.
Visualization of the expression cassette. It shows the order of the genes, an essential property due to the not 100% cleavage efficacy of the 2A self-cleaving peptides that link them.
Visualization of fMCS. This was extremely useful during the designing of primers for the Sanger sequencing of the unverified plasmid from Dr. Smith, as it allowed for the visualization of where in the sequence primers were, and approximately how close they were together. This representation also includes the region to be cut out and ligated with f3-55nm, as well as the restriction enzyme sites involved with that.
Visualization is of the final phage. This diagram includes the anti-CD22 scFv ligated into the pIII protein, as well as the expression cassette ligated into pAAV, and its subsequent removal along with the associated AAV2 sequences. It also includes all the sequence primers required for Sanger sequencing of the entire phage, along with insertion verification primers for the expression cassette.