Ryan David Ward ryandward

Protocol for Antibiotic Checkerboard Assay

I. Preparation of Antibiotic Serial Dilutions

Add 200µL of DRUG A (100x) and 300µL of LB into the top-right well of a deep well plate, yielding a 40x concentration in a total volume of 500µL.
Fill the rest of the wells in the top row with 250µL of LB.
Perform a serial dilution across the top row by transferring 250µL sequentially from one well to the next.
Discard 250µL from the penultimate well.
Repeat steps 1-4 in the bottom-left well using DRUG B instead of DRUG A.

Protocol for Library Growth with LB Media

Introduction

This protocol outlines the steps to grow up your libraries and perform chemical genomics for sequence quantification.

Materials

Library
Culture tubes
Eppitubes

Estimation of Bottleneck Size

The calculation of bottleneck size ($N_b$) follows the principle of effective population size estimation, adapted to quantify the 'founder' population size, i.e., the number of cells contributing descendants to the sample. The formula used in this study is taken from Krimbas and Tsakas and provides the best fit for estimating $N_b$ as per our data. It calculates $N_b$ using allele frequencies at initial and final time points ($f_{i_0}$ and $f_{i_s}$), the total number of sequence reads at both times ($s_0$ and $s_s$), and the number of generations.

This method is an attempt to formalize the intial development from this GitHub repository, which is based on the methodologies from this Nature Methods paper.

Shorthand	Strain	Notes	Paper	Table
Gallagher ... Manoil	5075	Most popular transposon library in Abau. References: Wang ... Mandel, Hauser	Gallagher et al (2015)	Presence means candidacy
Geisinger ... Opijnen, Isberg	17978	Best coverage; Tn chemical genomics	Geisinger et al (2020)	Column F
Wang ... Mandel, Hauser	17978	Poor coverage/sequencing/alignment; Tn-seq	Wang et al (2014)	[Tab A](https://journals-asm-org.ezproxy.library.wisc.edu/doi/suppl/10.

Lambda Red Recombination Protocol with sacB Selection

Overview

This protocol outlines the steps to prepare competent E. coli cells harboring the pSIM plasmid for lambda Red recombination using sacB as a counter-selectable marker. The process spans three days and includes streaking, culturing, induction of lambda Red proteins, cell preparation, transformation, and selection.

	= Arch Linux step-by-step installation =
	= http://blog.fabio.mancinelli.me/2012/12/28/Arch_Linux_on_BTRFS.html =


	== Boot the installation CD ==

	== Create partition ==

	cfdisk /dev/sda
	* Create a partition with code 8300 (Linux)

	#!/usr/bin/env bash

	function get_headphones_index() {
	echo $(pacmd list-cards \| grep bluez_card -B1 \| grep index \| awk '{print $2}')
	}

	function get_headphones_mac_address() {
	local temp=$(pacmd list-cards \| grep bluez_card -C20 \| grep 'device.string' \| cut -d' ' -f 3)
	temp="${temp%\"}"
	temp="${temp#\"}"