Merge pull request #48 from UBC-iGEM/Tina

Initial Thermostable TdT and ssDNA to dsDNA plans, #36

.local/share/rippkgs-index.sqlite

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+/etc/replit/rippkgs-indices/unstable.sqlite

.replit

@@ -1,8 +1,14 @@
 run = "mdbook serve -n 0.0.0.0"
+entrypoint = "main.sh"
+modules = ["bash:v1-20231215-e6d471c"]
 
 [nix]
 channel = "unstable"
 
 [[ports]]
 localPort = 3000
 externalPort = 80
+
+[deployment]
+run = ["bash", "main.sh"]
+deploymentTarget = "cloudrun"

src/SUMMARY.md

@@ -9,10 +9,9 @@
     - [March](./wet-lab/notebook/march.md)
 - [Design](./wet-lab/design/index.md)
   - [Solid phase DNA Synthesis](./wet-lab/design/solid-phase.md)
-  - [ssDNA to dsDNA](./wet-lab/design/ssdna-dsdna.md)
-  - [Thermostable TdT](./wet-lab/design/tdt.md)
+  - [ssDNA to dsDNA](./wet-lab/design/dsdna-experiment.md)
+  - [Thermostable TdT](./wet-lab/design/tdt-experiment.md)
 - [Protocols](./wet-lab/protocols/index.md)
-  - [TdT Cloning](./wet-lab/protocols/cloning.md)
 - [Engineering Success](./wet-lab/engineering/index.md)
 - [Parts](./wet-lab/parts/index.md)
 - [Results](./wet-lab/results/index.md)

src/bibliography.bib

@@ -408,3 +408,13 @@ @article{outlook_microfludics
   volume          = 3,
   year            = 2022
 }
+
+@Comment TdT Experiment 
+@article{TdT_background,
+  url             = {https://doi.org/10.1529/biophysj.105.074104},
+  author          = {Liang, X., Kuhn, H., & Frank-Kamenetskii, M. D.},
+  journal         = {Biophysical Journal},
+  pages           = {2877–2889},
+  title           = {{Monitoring single-stranded DNA secondary structure formation by determining the topological state of DNA catenanes}},
+  volume          = 90,
+  year            = 2006}

src/wet-lab/design/dsdna-experiment.md

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+# ssDNA to dsDNA Experimental Design
+
+## Overview
+
+Convert ssDNA synthesized by thermostable TdT (mutant 3-2) to dsDNA to generate a more stable dsDNA to allow better long-term storage.
+
+## Context and Scope
+
+Terminal deoxynucleotidyl transferase (TdT) is a special type of polymerase found in mammals that is able to synthesize ssDNA in a template-independent manner. Our project aims to utilize TdT to customize and synthesize DNA strands for data storage.
+
+Since TdT’s ability is limited to ssDNA synthesis, while ssDNA is not a stable biological molecule, this constrainsts our ability to store data long term using ssDNA. Hence, we propose to convert ssDNA to dsDNA once it is synthesized by TdT.
+
+## Goals
+
+- Synthesize dsDNA from ssDNA to achieve a more stable biological molecule for storage
+
+## Proposed solutions and workflow
+
+**PCR**
+
+1. Denaturation (~96°C):
+   Melt secondary structure, linearize ssDNA
+
+2. Annealing (~50-56°C):
+   Binding of primer to ssDNA template
+
+3. Extension (~72°C):
+   Taq polymerase extends the primers → dsDNA
+
+Primer design
+
+Primer is designed to be complementary to the initiator DNA of ssDNA synthesis and polyA tail
+
+Primer design requirement
+
+- 40-60 GC%
+- The forward and reverse primer can’t have a temperature difference of greater than 5C
+- 15-25 nucleotides long
+- Usually the melting temperature of the primer is 50-60C
+- Avoid hairpin structure
+
+## Plasmid integration
+
+Use cloning technique to integrate dsDNA into a plasmid in E.coli (PCR amplification, golden gate/Gibson assembly)
+
+Depending on the vector we use:
+
+- If we use type II restriction enzyme:
+
+PCR amplify the restriction enzyme recognition site onto the donor sequence (dsDNA) then perform Golden Gate to digest and ligate the dsDNA into the plasmid in E.coli in a one-pot reaction
+
+- If we use normal restriction enzyme:
+
+1. PCR amplify complementary region of the plasmid sequence onto the donor sequence
+
+2. Digest the plasmid with restriction enzyme
+
+3. Perform Gibson Assembly to ligate the dsDNA into the plasmid in E.coli
+
+**Transformation**
+
+Integrate the plasmid into E.coli BL21 (DE3) from NEB for protein expression
+
+**Colony Picking**
+
+Pick colony with correct antibiotic resistance
+
+## How do we test this?
+
+Options to see if this worked
+
+1. Use the same PCR primer to amplify the dsDNA inside the plasmid then use agarose gel to measure the length of the sequence integrated
+2. Sanger sequence or NGS (depending on the situation)
+
+## How long will this take?
+
+If everything goes wellwhile, 1-2 day. If not, 1 week should be enough for troubleshooting.

src/wet-lab/design/tdt-experiment.md

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+# Thermostable TdT
+
+## Overview
+
+The document provides the details related to Terminal deoxynucleotidyl transferase (TdT) utilized in the project. This includes the general description of WT TdT and thermostable TdT, in addition to their application in the project.
+
+## Context and Scope
+
+TdT is a specialized DNA polymerase that catalyzes the addition of nucleotides to the 3' terminus of a DNA molecule in a template-independent manner. This means it is able to synthesize single-strand DNA without an existing DNA strand as a template. This highlights its potential to be utilized as a biological tool to manipulate DNA synthesis, producing DNA strand as designed.
+
+ssDNA is prone to secondary structure formation (1). This issue can be minimized when working under a higher temperature (1). Since wildtype TdT derived from mammals cannot function optimally under a higher temperature (>37°C), this reaction can be achieved using a thermostable TdT, which has a higher optimal activity temperature, which in our case, would be around 47°C.
+
+## Goals
+
+- Clone thermostable TdT
+- Produce and purify thermostable TdT
+- Optimize TdT reaction condition
+
+## Design
+
+**Thermostable TdT Cloning: Ligation Independent Cloning (LIC)**
+Material:
+
+- Plasmid of Choice: Addgene # 29659 - pET His6 sumo TEV LIC cloning vector
+- Mutant TdT (purchased from IDT)
+- BL21 (DE3) (puchased from NEB)
+
+Procedure:
+
+1. Primer Design
+
+   - Design forward and reverse primer for TdT on geneious
+
+2. PCR Amplification
+
+   - Amplify the LIC fusion tag onto TdT sequence
+
+3. Agarose Gel
+
+   - Gel purification and check if the correct base pairs sequence has been added onto TdT
+
+4. Linearize the plasmid
+
+   - Use SspI restriction enzyme to linearize the vector
+
+5. Gel Purification
+
+   - Gel purify the linearized product and if needed the PCR product
+
+6. Ligation Independent Cloning
+
+   - Use T4 DNA Polymerase reaction with dCTP for insert and dGTP for vector
+
+7. Transformation
+
+   - Transform chemically competent Top10 e.coli with cloning product
+
+8. Colony picking
+
+   - Pick Kanamycin-resistant colonies
+
+9. Miniprep
+
+   - Use Miniprep to isolate plasmid from E.coli
+
+10. Sequencing
+
+    - Send plasmid for Sanger sequencing
+
+**Protein Purification: Immobilized metal affinity chromatography**
+
+Protocol reference [link](https://link.springer.com/protocol/10.1007/978-1-59745-582-4_2)
+
+**TdT Optimization**
+**Liquid Phase (Gel)**
+
+- Each reaction was carried out in 20µL total volume.
+- All reaction components but the dNTP were assembled in 18µL
+  dNTP was prepared in 2µL of water.
+- The 18µL mix was composed such that upon mixing with the 2µL dNTP solution, the following initial composition would be obtained: 200µM dNTP, 1X TdT buffer, 0.05µM primer (TBD) 250µM cobalt chloride (if present), 1U/µL TdT
+- To initiate the reaction, the 18µL mixture was added to a tube containing the 2µL dNTP mix and mixed immediately by pipetting.
+- The reaction was then incubated at room temperature for at least two minutes, resolved on a TBE Polyacrylamide gel
+- Length of ssDNA is determined by comparing with the ladder
+
+**Condition to compare:**
+
+Run reaction with each dNTP + ladder + primer reference
+
+- Different dNTP concentration: 10, 25, 50, 100, 200, 400µM
+- Different TdT concentration
+- Different buffer concentration
+- Different temperature: RT vs 37 vs 47 (mutant)
+- With/without CoCl2: 0 vs 2.5mM vs 5 mM
+- Different reaction time: 2 vs 10 vs 30 min
+
+Testing dNTP concentration need for all 4 types of nucleotides
+The rest rxns can be carried with selected dNTPs
+
+Protocol reference [link](https://www.nature.com/articles/s41467-019-10258-1#MOESM1)
+
+**Solid Phase (TBD)**
+
+## How long will this take?
+
+This depends heavily on how successfully each experiment goes. The estimation is around 1 month.
+
+[@TdT_background]