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Everything to do with the Mur Ligase Project
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AZ Accumulation Assay #113

Open Yuhang-CADD opened 2 months ago

Yuhang-CADD commented 2 months ago

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:

Screenshot 2024-07-23 at 10 20 13

Calculator

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

Yuhang-CADD commented 1 week ago

Biological Part Experimental:

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

Procedure:

Screenshot 2024-09-18 at 17 44 37 Screenshot 2024-09-18 at 17 44 53 Screenshot 2024-09-18 at 17 45 06 Screenshot 2024-09-18 at 17 45 19 Screenshot 2024-09-18 at 17 45 29

Materials:

Screenshot 2024-09-18 at 17 43 16 Screenshot 2024-09-18 at 17 43 31

Equipments

Screenshot 2024-09-18 at 17 42 34

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)

Yuhang-CADD commented 1 week ago

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