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| | {{:Team:Arizona_State/Templates/sidebar|title=Notebook: May}} | | {{:Team:Arizona_State/Templates/sidebar|title=Notebook: May}} |
| - | __NOTOC__
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| - | == Thursday, May 5 ==
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| - | * Pitching our CRISPR idea to Xiao
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| - | * Needs to be more accessible (how can we explain CRISPR to a non biologist?)
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| - | == Wednesday, May 11 ==
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| - | * Leader sequence- conservation, what activates (link to HN repression paper)
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| - | :* We know it is AT rich
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| - | :* Can we insert our own promoter?
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| - | * How robust is CRISPR?
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| - | * We need a proof of concept- GFP silencing
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| - | :* Will need 2 plasmids since CRISPR will probably linearize any target
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| - | :* Signal intensity correlated with expression
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| - | :* "Repress the repressor"
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| - | :* As vanilla as possible
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| - | * How can we be compatible with the Biobrick standard?
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| - | * What happens if we insert our own CRISPR plasmid into a cell with a native system?
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| - | == Thursday, May 12 ==
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| - | * Problems with targeting essential genes- endogenous CRISPR inserts are selected against
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| - | :* Any resistance targeting will be heavily selected against
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| - | :* Only laboratory applications?
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| - | * Modeling ideas:
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| - | :* Target vital genes- how does the cell respond?
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| - | :* Look at rate of transmission during replication
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| - | :* Population dynamics- how is CRISPR array transferred throughout a colony of cells? After a phage challenge?
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| - | * What is our end game with CRISPR? Realistically what could it be used for in the real world?
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| - | == Monday, May 16 ==
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| - | * Talking about leader sequence- we should be able to insert our own lac promoter to express the CAS genes and the CRISPR array.
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| - | == Tuesday, May 17 ==
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| - | * Finished semi-official proposal for project
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| - | * Some form emails
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| - | * How to construct our plasmids:
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| - | :* PCR our CAS genes incorporating appropriate restriction sites
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| - | :* Get the array synthesized?
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| - | == Wednesday, May 18 ==
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| - | * How many repeat / spacer units? What do we need to take into account when deciding this?
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| - | * How can we make our design modular and easily customizable?
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| - | * Synthesize an array with 3 repeats and restriction sites at spacer locations?
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| - | * Which part of GFP gene do we put in our 32bp spacers? Does it matter?
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| - | * Are we targeting DNA or RNA?
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| - | :* DNA: CAS, much more literature = we know more about the mechanism = greater chance of success
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| - | ::* 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.
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| - | ::* "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."
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| - | :* RNA: RAMP, only a few papers in the literature about this (link?), not in very many organisms
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| - | == Friday, May 20 ==
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| - | * Xiao came in this morning, and today we are joined by Abhinav, a BME undergrad in his lab
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| - | * DNA method: we can examine the fluorescence curve (flow cytometry) when we add in the GFP plasmid.
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| - | :* 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
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| - | * Backups: we need to have more genes to target than just NDM-1
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| - | :* Multiple: target a cluster of genes that code for parts of a characteristic
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| - | ::* To inhibit the entire characteristic, we would have to silence A, B, and C (or something like this)
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| - | * Other ideas:
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| - | :* Motility (csgA)
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| - | :* Synechocystis (cyanobacteria that is big at ASU)
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| - | :* Biobrick with AND gate (need 1-1)
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| - | :* Origin of replication, prevent horizontal gene transfer
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| - | == Tuesday, May 31 ==
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| - | * First official day in the lab
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| - | * Received two strains of E. coli from life sciences
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| - | :* (strain info here)
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| - | :* 1 strain with no native CRISPR system
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| - | * Made 4 plates from this source plate (2 each strain)
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