Extraction of pBBR1-MCS5-yedQ plasmid

Genomic DNA from E. coli MG1655 and primer design

Amplification of linearized pBBR1-MCS5-yedQ (Y2, Y3) and P_fnrS (F1, F2, F3) fragments

Amplification of linearized pBBR1-MCS5-yedQ (Y1)

Gel electrophoresis to verify the 3.4 PCR products and purification

Primer Design

Gibson assembly and transformation

Gibson assemblies (Y2-F2 and Y3-F3) and transformation

Colony PCR

Plasmid extraction from the 9 colonies picked on 3.10

PCR and gel electrophoresis for the 9 plasmids extracted on 3.11

Designed verification primer set 2

PCR of the 9 plasmids using primer set P_fnrS-certify2-FR

Repeat PCR using new reagents

pBBR1-MCS5-yedQ plasmid certify

Colony PCR

pBBR1-MCS5-yedQ-P_fnrS plasmid

Bacterial resuscitation and Microaerobic Aerobic culture

ELISA validation of c-di-GMP production

Bacterial resuscitation

ELISA

Bacterial resuscitation and culture under Aerobic, Microaerobic, Anaerobic environments

ELISA and CFU measurement

Bacterial resuscitation and culture

ELISA

Bacterial Transformation of the pBBR1-MCS5 empty vector

Bacterial glycerol stock

Bacterial resuscitation and culture with IBMX

ELISA

Bacteria recover and Primer Design

Plasmid Extraction and Amplification for Vector Linearization and GmR Fragment

Repeat PCR for pUCP20 Backbone Linearization

Gibson Assembly for Cloning GmR Cassette into pUCP20

PCR Amplification of P_fnrS-yedQ Fragment

PCR Linearization of pUCP20-GmR Vector

Re-optimized PCR for pUCP20-GmR Linearization

Next-Generation Sequencing of Linearized pUCP20-GmR

Gibson Assembly for Final Construct

Colony PCR Screening for Positive Clones

ELISA verification (pUCP20-GmR Vector)

Primer Design

PCR for Linearization and fragments

DNA Purification

Gibson Assembly Reaction and Bacterial Transformation

Verification of Clones

Sequencing

PCR Repeated

Gel Extraction and Gibson Assembly

Verification of Clones again

Colony PCR and Sequencing

Bbacterial Glycerol Cryopreservation

Bacterial Culture Setup

Measurement

Data Analysis

Strain Revival and Plasmid Extraction

Heat Shock Transformation

Colony PCR

Growth Curve Assay

Plasmid Construction Workflow

Data Acquisition and Viable Plate Count

Validation by Agar Diffusion Assay and spot plate assay

Supplement

Bacterial Culture

Plasmid Extraction

Colony PCR of double-plasmid strain identification

Strain cryopreservation

Co-transformation and Colony PCR identification

Inoculation and Biofilm Formation

Crystal Violet Staining

Data Analysis

E. coli Genomic DNA Extraction and Transformation

Colony PCR for E. coli(pKD46)

PCR Amplification of Target Fragments

Extract pKD46 plasmid

Electroporation

Transformation of pKD3 and pCP20

Electroporation and plasmid extraction

Picking Single Colonies and incubation

PCR Amplification of Target Gene Flanking Regions

Knockout Verification

Attempts to Remove Chloramphenicol Resistance Cassette from ΔthyA Strain Using pCP20 and Repeated Attempts to Knockout dapA

Bacterial Handling and cell culture

Sample Collection and detection at 4h

Sample Collection and detection at 22h

IFN-β ELISA for Frozen Supernatants

Data Processing

Raw data of the Flow-Cytometric 4h

Raw data of the Flow-Cytometric 22h