AZ Accumulation Assay

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Protocol Following:

https://www.nature.com/articles/s41596-021-00598-y

Sample List:

https://docs.google.com/spreadsheets/d/1LPFFPpNr6e6BsTX1GKkTgr84WCbT3ZSG47DjbhU2kxI/edit?gid=0#gid=0

Accumulation Assay Sample Preparation:

Calculator

https://www.sigmaaldrich.com/GB/en/support/calculators-and-apps/mass-molarity-calculator

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Biological Part Experimental:

{pre-announcement: screenshots taken from the original paper were used for fair academic discussions}

Procedure:

Materials:

Equipments

Biological Part Discussions:

General molecular biology lab can potentially do this assay. (all we need are: 37 Celsius degree shaker, Falcon tubes, OD600 spectrometer, centrifuges, liquid nitrogen kit, ultra pure water, and bacteria needed. Any other consumables (not mentioned) should be available for most general biolabs.

Tips on doing the assay:

1. don't miss the exact timing to avoid bacteria overgrowth (incoherence in growth curve), OD=0.55-0.6 is very good range! Otherwise, the bacterial concentration will be different and health and accumulation will be different upon overgrowth

2. On usage of falcon tubes (cheap, available in stores, don't overfill, less than 1/3 of the volume to leave enough pressure to avoid pop-off upon heating and shaking, and leave enough air for bacterial growth) plus, the cap loosen for more air to come in and less chance of popping off upon shaking

3. don't forget to set the high and low accumulation controls for the assay (purchase in advance if necessary)

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Mapping against Eco, Pae (efflux-, penetration-weakened, WT)

Firstly need to test against WT Eco & Pae Can also test our newly designed AZ derivative compounds against Eco TolC- or Pae MexABCDXY- strains for further guide on influences of permeation barrier Same rationale in testing compounds against Colistin pretreated Eco/Pae strains for further guide on efflux pumps' influence. https://pubs.acs.org/doi/10.1021/cb500360c

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Grid of compounds to be tested (Round 1):

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Brief Summary: Eco (K12 non-resistance developer) ready to do this year (from mid Nov) but will take a bit of time for resynthesis & paperwork for chemical transfer via institutes (will update soon) Eco TolC- & Pae next year

Sample screening time: super fast for 20 compounds (estimated 2 weeks max for chip-ins) Sample requirement: 10 mM (100 uL) DMSO, as clean as possible (HRMS),

Prefer to screen one by one

Can do MIC quickly as well.