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Notebook: May

Thursday, May 5

• Pitching our CRISPR idea to Xiao
• Needs to be more accessible (how can we explain CRISPR to a non biologist?)

Wednesday, May 11

• Leader sequence- conservation, what activates (link to HN repression paper)

• We know it is AT rich
• Can we insert our own promoter?

• How robust is CRISPR?
• We need a proof of concept- GFP silencing

• Will need 2 plasmids since CRISPR will probably linearize any target
• Signal intensity correlated with expression
• "Repress the repressor"
• As vanilla as possible

• How can we be compatible with the Biobrick standard?
• What happens if we insert our own CRISPR plasmid into a cell with a native system?

Thursday, May 12

• Problems with targeting essential genes- endogenous CRISPR inserts are selected against

• Any resistance targeting will be heavily selected against
• Only laboratory applications?

• Modeling ideas:

• Target vital genes- how does the cell respond?
• Look at rate of transmission during replication
• Population dynamics- how is CRISPR array transferred throughout a colony of cells? After a phage challenge?

• What is our end game with CRISPR? Realistically what could it be used for in the real world?

Monday, May 16

• Talking about leader sequence- we should be able to insert our own lac promoter to express the CAS genes and the CRISPR array.

Tuesday, May 17

• Finished semi-official proposal for project
• Some form emails
• How to construct our plasmids:

• PCR our CAS genes incorporating appropriate restriction sites
• Get the array synthesized?

Wednesday, May 18

• How many repeat / spacer units? What do we need to take into account when deciding this?
• How can we make our design modular and easily customizable?
• Synthesize an array with 3 repeats and restriction sites at spacer locations?
• Which part of GFP gene do we put in our 32bp spacers? Does it matter?
• Are we targeting DNA or RNA?

• DNA: CAS, much more literature = we know more about the mechanism = greater chance of success

• How do we get GFP to stay in the cell if we target it with CRISPR? Use a separate plasmid, but without antibiotic selection mechanism the cells will die.
• "You think of it as a happy GFP story...but really there's a lot of whipping going on. You kill the whole bacterial family until you just get the GFP ones."

• RNA: RAMP, only a few papers in the literature about this (link?), not in very many organisms

Friday, May 20

• Xiao came in this morning, and today we are joined by Abhinav, a BME undergrad in his lab
• DNA method: we can examine the fluorescence curve (flow cytometry) when we add in the GFP plasmid.

• If it takes a while to decrease, we can then examine what it looks like when CRISPR is added and have a point of comparison. However, if it attenuates quickly, this isn't really an option for us

• Backups: we need to have more genes to target than just NDM-1

• Multiple: target a cluster of genes that code for parts of a characteristic

• To inhibit the entire characteristic, we would have to silence A, B, and C (or something like this)

• Other ideas:

• Motility (csgA)
• Synechocystis (cyanobacteria that is big at ASU)
• Biobrick with AND gate (need 1-1)
• Origin of replication, prevent horizontal gene transfer

Tuesday, May 31

• First official day in the lab
• Received two strains of E. coli from life sciences

• (strain info here)
• 1 strain with no native CRISPR system

• Made 4 plates from this source plate (2 each strain)