Notebook

Detection Module
Extracted E.coli genome
Searched sequences and designed primers
Function Module
Prepared kanamycin culture medium
Prepared kanamycin and chloramphenicol medium
At the same time, introduced empty pET-28A(+) plasmid and nirB/icd plasmid as negative control

Detection Module
Amplified nirB and icd sequences fragments via PCR
Gibson assembly with corresponding fluorescent protein (nirB mRFP, icd eGFP) and loading into p5T7 plasmid
sent for sequencing
Transformation of recombinant plasmid harboring mRFP into E. coli
Function Module
Recombination of mRFP with Escherichia coli
Resistance screening of kanamycin and chloramphenicol, liquid medium culture
Detected the change of fluorescence intensity with time-variant and ploting A1
Detected the change of OD (bacterial count) with time-variant and draw B1
Preparation culture medium contains 5 μ M hemin
Introduction of recombinant plasmid of leghemoglobin

Detection Module
Designed primers for the amplifaction of fragments including nirB, eGFP, mRFP and icd, and interted them into plasmid P5T7
Scribing of mRFP Escherichia coli transformation
Function Module
Kanamycin resistance screening and liquid medium culture
Group 1: No hemin culture
Detection of expression effect by SDS-PAGE: C21
Detect the change of OD (bacterial count) with time-variant and draw B2
Detect the change of fluorescence intensity with time-variant and draw A2
Group 2: Add 5 μ M hemin
Detection of expression effect by SDS-PAGE: C22
Detect the change of OD (bacterial count) with time-variant and draw B2
Detect fluorescence intensity and OD (bacterial load) over time and plot A22 and B22.

Detection Module
Culture bacteria and Extract plasmid
Two plasmids of nirB+mRFP and icd+eGFP were obtained by PCR using P5T7 plasmid as the vector
DNA fragments recovery
Design and verify primers
Culture bacteria and Extract plasmid
Function Module
construction of plasmid with laccase carrying signal peptide
Introduction of recombinant plasmid carrying signal peptide laccase
Kanamycin resistance screening and liquid medium culture
SDS-PAGE is used to detect the expression effect C3

Detection Module
Carried out strain PCR, scribing and Gibson's large intestine transformation to obtain the required plasmid
Scribed and added strain PCR to verify correctness
Culture bacteria.
Extracted the plasmid, and found the concentration is normal,which between 100 and 200 ng
Culture bacteria and Genome extraction
Design sequencing primer (about 1kb) and send it to the company for detection
Function Module
Introduction of signal peptide laccase plasmid and detection plasmid
Screening with kanamycin and chloramphenicol
Detect the change of fluorescence intensity with time-variant and draw A3
Detect the change of OD (bacterial count) with time-variant and draw B3

Detection Module
Through sequencing results, analyzed and judged whether the plasmid construction was successful to verify whether the interface is correct
Culture bacteria and Extract plasmid
Determined the culture conditions, as well as the subsequent test forms and data, and analyzed the data
Design the evacuation unit and determine which two specific gases to use
Determine the volume ratio of the gas to be charged and how long to incubate to start testing
Import the plasmid including nirB+mRFP
Culture nirB plasmid under two media
Function Module
Detect the change of fluorescence intensity with time-variant and draw A31
Detect the change of OD (bacterial count) with time-variant and plot B31
Introduction of laccase recombinant plasmid without signal peptide
Kanamycin resistance screening and liquid medium culture
SDS-PAGE to detect the expression effect C4

Detection Module
Configure normal medium without cysteine
Configure hypoxic medium with cysteine
Culture the two plasmids under normal medium
Sample the plasmids of both assay modules cultured on normal media at regular intervals
Culture the two plasmids under hypoxic medium
Sample the plasmids of both assay modules cultured on hypoxic medium at regular intervals
Measure the absorbance and fluorescence intensity of each time gradient by enzyme marker
Function Module
cotransformation of leghemoglobin plasmid and CueO plasmid, noted as pcl-group
Add 0.2 mmol/L copper ions to CueO+nirB+Lb and add IPTG after 3h
Detect fluorescence intensity changes over time and plot A71
Detect the change of OD (bacterial load) with time and plot B71
Add 0.5 mmol/L copper ions to CueO+nirB+Lb and add IPTG after 3h
Detect fluorescence intensity changes over time and plot A72
Detect the change of OD (bacterial load) with time and plot B72