Overview

This page contains a list of materials (bacterial strains, plasmids, primers, antibodies) and methods for this project. Original source material(s) for the protocols are indicated where relevant.

Materials

Bacterial Strains

Strain	Description	Source
E.coli K12	Standard laboratory strain	Mutations Lab, IISER TVM
E.coli DH5ɑ	Strain optimized for cloning	LICB, IISER TVM
E.coli Mach1	Fast growing, chemically competent cells	Host-Pathogen Interaction Lab, IISER TVM
E.coli XL10 gold	Ultracompetent, fast growing competent cells	Mutations Lab, IISER TVM
E.coli K12 ΔtolA	K12 strain with gene knockout ΔtolR::Kan	Keio collection
E.coli K12 ΔTolR	K12 strain with gene knockout ΔtolA::Kan	Keio collection
E.coli K12 Δnlp1	K12 strain with gene knockout Δnlp1::Kan	Keio collection
E.coli K12 ΔdegP	K12 strain with gene knockout ΔdegP::Kan	Keio collection

Plasmids

Plasmid	Description	Source
pGEX-4T1	Plasmid for protein expression in E.coli. pBR322 origin, IPTG inducible tac promoter	Dr. Vipul Gujrati, TUM Germany
pET-28a	Plasmid for protein expression in E.coli. IPTG inducible T7 promoter	Mutations Lab, IISER TVM

Primers

Primers	Description	Sequence
ClyA_Myc_Affi_FP1	Forward sequencing primer for ClyA-Myc-Affi (backbone specific)	cataattcgtgtcgctcaaggc
ClyA_Myc_Affi_FP2	Forward sequencing primer for ClyA-Myc-Affi (insert specific)	ccaaacagtgtatgaatggtgtgg
ClyA_Myc_Affi_RP1	Reverse sequencing primer for ClyA-Myc-Affi (backbone specific)	caagctgtgaccgtctcc
ClyA_THP_FP1_EcoNI	Forward primer for ClyA-3XFLAG-THP	TTGAACCCTATACTAGGCAAATTGATGGCCT
ClyA_THP_RP1_BamHI	Reverse primer for ClyA-3XFLAG-THP	GTTACGGATCCGATTTAACAAAACG
ClyA_Affi_FP1_EcoNI	Forward primer for ClyA-V5-Affi	ttgaacCCTATACTAGGAaagctaatg
ClyA_Affi_RP1_BamHI	Reverse primer for ClyA-V5-Affi	GTTACggatccAATtcattttggtg

Antibodies

Antibody	Catalog number/supplier	Dilution used
Anti OmpA Ab E. coli	Antibody Research Corporation, Prayaga Scientific	1:200 (Cell culture) 1:300 (Western blot)
Anti V5-Tag, rabbit mAb	#13202, Cell Signaling Technologies	1:400 (Cell culture) 1:1000 (Western blot)
ANTI-FLAG M2, Clone M2, mouse mAb,	Sigma-Aldrich	1:300 (Cell culture)
Alexa Fluor™ 488 conjugated AffiniPure Goat anti-Rabbit IgG (H+L)	Jackson ImmunoResearch laboratories	1:1000 (Cell culture)
Alexa Fluor™ 488 conjugated AffiniPure Goat anti-Mouse IgG (H+L)	Jackson ImmunoResearch laboratories	1:1000 (Cell culture)
Cyanine Cy™ 5 conjugated AffiniPure Goat anti-Rabbit IgG (H+L)	Jackson ImmunoResearch laboratories	1:1000 (Cell culture)
Anti-myc tag primary antibody, Rabbit	Thermo Fisher	1:500 (Western blot)
Anti-Rabbit HRP conjugated antibody	Antibody Research Corporation	1:5000 (Western blot)
V5-Tag, rabbit mAb	#13202, Cell Signaling Technologies	1:400 (Cell culture) 1:1000 (Western blot)

Softwares

Software	Use
R Studio	Analysing growth curve and Bradford assay results
Graphpad Prism	Plotting and visualising results
MATLAB	Math modeling
Python	Math modelling
ImageLab	Imaging and analysing SDS PAGE gels and Western blots
A Plasmid Editor	Analysing DNA sequences
SnapGene	Analysing DNA sequences, designing primers, analysing chromatograms

Methods

Bacterial Cultures

Cloning

OMV Isolation and Characterization

Protein Quantification and Identification

Mammalian Cell Culture and Cellular Uptake Experiments