READ ME File For 'Dataset in support of the Southampton doctoral thesis'

Dataset DOI:  https://doi.org/10.5258/SOTON/D2425

ReadMe Author: Emma Cousins, University of Southampton ORCID ID: https://orcid.org/0000-0002-6962-3797

This dataset supports the thesis entitled Studies on plasmid-based genetic tools for C. muridarum: progress towards a replicating vector for transposon mutagenesis.
AWARDED BY: Univeristy of Southampton
DATE OF AWARD: 2022

DESCRIPTION OF THE DATA

Plasmid-loss was measured by two independent methods. Inocula for individual C. muridarum strains was serially passaged three times (P1-3) either with or without selection with chloramphenicol. Inocula harvested at P3 was passaged three times either with or without selection with penicillin. Bacteria were harvested from cultures in one T25 culture flask at 28 hpi and stored in 4SP at -70˚C. Harvested EBs at each passage represent individual samples numbered. The concentration of infectious forming units (IFU ml-1) were calculated for each sample by titration by Chlamydia binding genus-specific LPS monoclonal antibody (Mab29) which were stained with X-gal. Titres were used to calculate the volume required to infect a T25 culture at MOI=1 at each passage. 
Plasmid copy number (CDS2: omcB) ratios were determined for samples at P3 and P6 by qPCR. the total genomic DNA was extracted from samples at passage 3 and 6 only for determination of plasmid copy number (absolute plasmid copies per chromosome) by qPCR which used primer pairs to target single copy genes located on the chromosome (omcB) and on the plasmid (CDS2). Quadruplicate technical repeats were performed for each sample and the DNA standards were run in duplicate. All samples for Nigg P-/pGFP::Nigg were run on the same plate requiring one standard curve for both the plasmid and chromosome assays. All samples for Nigg P-/pSW2NiggCDS2, Nigg P-/pNiggCDS56Del and Nigg P-/pNiggCDS567Del were run on the same plate requiring one standard curve for both assays.


Phenotypic analysis was performed on images of cell cultures at each passage to quantify the frequency of plasmid loss as a proxy for the rate of plasmid loss, by quantifying the proportion of plasmid-free inclusions in the total population identified by the absence of GFP expressed in trans. The proportion of  inclusions that did not express GFP from the transforming plasmid (plasmid-free Chlamydia) in the total population for each culture was determined from two fields imaged for each culture flask. For all images of C. muridarum transformants that fluoresced green in cell culture, the total number of inclusions within a McCoy cell monolayer was automatically quantified using Image J™ software version 1.53k. Images were converted into black and white and then to 8-bit resolution. The minimum and maximum threshold was set from 20 to 255 respectively with a dark background selected. To allow the software to detect individual inclusions that were so close in proximity that they did not appear as separate inclusions, the watershed function was selected to add a separating line of 1 pixel in diameter between each inclusion. The pixel size was set to 700-10,000 for analysing particles (a particle is the same as one inclusion in this instance) that fell within this range. The ability of this size range to detect all mature inclusions was verified by manual counts of the same images, performed for two images. The circularity was set from 0.1 to 1.00 where 0.0 is a straight line and 1 is a complete circle. Inclusions that did not flouresce green were counted manually from the same images.


Samples labelled A and B are biological repeats of the same experiment and so are samples labelled C and D.  


This dataset contains:
The data for plasmid copy number is presented in a table in an Excel Spreadsheet and shows the raw qPCR data (absolute counts of copy numbers for chromosomes and plasmids), the average copy number of four technical repeat measurements for two biological repeats for each strain and the ratio of plasmids to chromosomes for each strain. 
The data for plasmid loss frequency is presented in a table in an Excel Spreadsheet and shows The total inclusion counts and the total number of plasmid-free inclusions counted for duplicate technical repeats for duplicate samples. 


Date of data collection: 01.10.2017-30.09.2021

Information about geographic location of data collection: 

Licence:
Creative Commons Attribution CC-BY


Date that the file was created: October, 2022

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