August 5th
@Yujeong Oh @Adrian Villanueva @Antonina Bar
Presentation:
Notes:
- Gene of choice
- ITS region; need approval from Hussain to test in the lab because this contains the coding part
- email him a short paragraph explaining what ITS region is and overall plan for our project. Ask for approval whether we can test this in the lab
2. Reporting mechanism
- Fluorescence: Combine prof Song's fluorescence concentrator in our device
- Ibrahim suggested one person from the engineering to work on this
3. Fall semester Lab work plan & suggestion
*Suggestions
- Gene fragment (gBlock, our target gene)
- instead of ligated into pJET vector (blunt-end ligation)
- research on one-step insertion of the gene fragment (gBlock) into the E. Coli
- Request meeting with Prof Youssef or his postdoc/ researcher
- they are working on detection of COVID using fluidigm on RT-PCR
- more sensitive than governmental/ official testing
- ask him to share how they are doing
- ask for whether we can work with them/ their method on our few samples
- if allowed, order the reagents needed in their method
- Request meeting with Prof AJ
- ask him about nucleic acid detection
- we previously tried to contact him, but didn't receive an reply
*Lab plans & advices
- Specificity test: PCR/RPA/LAMP on miniprepped sample
- test on gel to check specificity of the primers
- Sensitivity test: PCR/RPA/LAMP on serial diluted miniprepped sample
- test it on gel and with SYBR green
- one might be more sensitive than others
- LAMP has high negative control contamination
- what are we detecting? DNA region vs. RNA region
- ITS region is DNA region.
- Do we need to work on RNA region?
- For working with RNA, high number of samples are needed. Sometimes, the researchers work with the plasmid that express the RNA
- work with BL21 E.coli strain (not DH-alpha) that express and transcribe DNA to RNA
- Due to time restriction and budget cut, Ibrahim suggested us to focus on working with DNA detection this year
- On wiki, we can list down the reseaches that we've done on detection of DNA and RNA.
- add in the wiki that this might be a 2 year project and the following RNA detection part will be done in wet lab the following year.
- Indeed, we can still experiment CRISPR-Cas13 using T7 transcription
- post amplifcation → T7 transcription → CRISPR-Cas13
- more research is required in this method
- Things to consider: CRISPR-Cas13 is more sensitive than Cas12. Is it important? Do the detection require that sensitivity? How about time taken including T7 transcription step?
- This should be discussed with faculties. Ibrahim strongly suggested us to have a meeting with faculties soon; if the general meeting cannot be scheduled, at least have a meeting from biology team.
4. Order
- $10,000 allocated for bio and engineering orders
- this is 1/6 of the previous year's order budget. This makes us harder to order all reagents that we want to order
- We went over the order sheet together and marked the uncertain orders as yellow
- If no payment is required for iGEM from now - september, place the order soon (within two days)
5. Future Plan
- Parts (medal requirement)
- our primers & gBlocks will be enough for new part characterization
- Add documentation to existing part: do literature review on 2-3 existing parts and find & add new information
- as a backup, Ibrahim suggested the engineering team to work on this criteria (i.e. design something for our project using 3D printer and take a video on how to make it).
- Cost anlaysis
- Computational analysis
- Real sample testing is worth it to test in lab
- To show our proof of concept (one of gold medal criteria)
- It will be also nice to integrate biology parts with the engineering device to show the proof of concept
- one person from biology team should work on the sample procurement
Next Steps:
*These are the next steps that I could think of after the meeting, please let me know if there are somethings else :)
- Email & Get approval from Hussain
- Research a method to insert the gBlock into E. coli DH-alpha plasmid without using pJET ligation
- Request meeting with Prof Youssef
- Request meeting with Prof AJ
- Research more on T7 transcription and SHERLOCK on DNA detection
- Schedule a faculty meeting to decide whether we will test SHERLOCK method
- Sample procurement from the German researcher
- Cost analysis
- Computational analysis
- Documentation on existing parts : literature reviews
- Follow up with Jeff & Place the order