Team:GZ HFI/Notebook

Notebook | iGEM GZ_HFI


Construction of ammonia metabolic pathway

  1. Construction of plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ^ fbr;

  1. Knockout the argR gene in E.coli DH10b;

  1. Transfer pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ^ fbr to E.coli DH10b ΔargR;

  1. Culture preservation

Week One (20200627-20200628)

  • ArgA wild-type was amplified from E.coli DH10b genome by PCR and connected to pSB1C3.


Name of cloneDH10B{pSB1C3-argA_EcoWT}pSB1C3-argA_EcoWT.dna
strain sourceargA From DH10B pSB1C3 From BluephalabpSB1C3-fwYellow.dna
MethodsPCR, Enzyme digestion and Ligase

The strain DH5 containing plasmid pSB1C3 was cultured

ResistanceChl (Cm)
Culture Condition:shaking tables37 ℃200 rpm


Preserve cultured strain DH5α containing plasmid pSB1C3-FwYellow for later use.

Strain preservation 001
Label nameHFI001
Source bacterial solution1
Preservation method50% glycerol solution 1: 1 miscible, stored at -80 ℃
Name of preservationDH5α{pSB1C3-FwYellow}

Extraction of plasmid pSB1C3-FwYellow from DH5α

Product NameHFI001 6.28
Source bacterial solution1
Plasmid namepSB1C3-fwYellow
Extraction methodRapid Extraction KitSample2mL
EluateddH2OElution volume50 μLconcentration121.4 ng/μL

Excision of the yellow protein gene fragment from the plasmid pSB1C3-FwYellow

Label nameHFI
Substrate DNApSB1C3-fwYellow
Total volume50 μLSubstrate amount10μL
Endonase (separated by semicolons)EcoRI;SpeI
BufferNEB Cutsmarttemperature37 ℃
Reaction duration60 minIs it inactivated?Yes.
Expected product length1.1 kb;2.0 kb”

Amplify the original ArgA gene from DH10b

Label nameargA
Template sourceDH10b
Primer (separated by semicolons)argA-F1;argA-R1
Total volume50 μL
Polymerase typeFastPfuTemplate QuantityColony
Annealing temperature55 ℃Extension time setting30 s
Expected product length1.3kb

Verify the proposed ArgA gene (validated correctly)

Agarose gel electrophoresis 005
Concentration0.01MarkerTrans8K Genestar 2000
Voltage130 VTime25 min
Sample loading6 μL
Layout descriptionLane1:PCR004; Lane2:003; Lane3:pSB1C3-fwYellow; Lane4:2K Marker; Lane5: Trans8K

Week Two (20200629-20200705)

  • Construction of argA fbr

  • Sequencing


To retrieve the remaining ArgA gene.

Product NameargA
Sample Source0628【15:00】argA
Extraction methodTiangen universal DNA recovery kit
Elution volume50 μLconcentration38.4 ng/μL
RemarksStore the recovered product in 4 degree refrigerator blue board


Modify the ArgA gene to have the same sticky end as the plasmid.

Enzyme digestion 007
Label nameF-HFI
Substrate DNADNA fragment recovery 006
Total volume50 μLSubstrate amount25μL
Endonase (separated by semicolons)EcoRI;SpeI
BufferNEB Cutsmarttemperature37 ℃
Reaction duration60 minIs it inactivated?
Expected product length1.3kb
RemarksAfter digestion and inactivation, directly used for T4 link


The product mixture was obtained by linking ArgA gene with plasmid digestion system by PCR.

T4 Connection 008
Label name1C3-argA
Total system20μL
Fixed component (automatic calculation)
10× T4 ligase bufferμL
T4 ligaseμL
0630 F-HFI2μLNo resistance
0628 HFI (vector)1μLResistance marker
ddH2O14μLPhosphorylated fragment

The product mixture was transferred into receptive cells, cultured, and unluminescent positive monoclones were found and cultured.

Tablet NamepSB1C3-argA_EcoWT
Transform DNA sources.T4 Link 008
DNA addition10μL
Sensitive cellEPS300
Sensitive state usage50μL
Resuscitation mediumLB300μL
Coated plate9 cm板LB
Resistance markerChl
Coating amountAfter centrifugation
RemarksNegative clone with fluorescence


Inoculation 010
Name of bacteria0701 transform to 009
Resistance (multiple)Chl (Cm)
From:LB colony plateTake:3Colony
Dilute to5mLLB LiquidTest tube
Culture conditions:Shaker37 ℃200 rpm
RemarksFor plasmid extraction

Colony PCR, replication positive monoclonal

Template sourceInoculation 010
Primer (separated by semicolons)F; 2R
Template quantity (each)1Colony
Number of repackaged tubes (about 0.5 more tubes are recommended than expected):6Volume per tube20μL
Total system preparation (automatic calculation):
Total preparation120μL
2×Taq Mix60μL
Primer 16μL
Primer 26μL
Annealing temperature55 ℃Extension time setting100 s
Expected product length1.6kb


Extraction of plasmid

Plasmid extraction 012
Product NameEC3-ArgA 1 ~ 3
Source bacterial solutionInoculation 010
Plasmid nameArgA
Extraction methodRapid Extraction KitSample2mL
EluateddH2OElution volume50 μLconcentration100 ng / μL
RemarksConcentration measurement for nanoDrop


Using PCR mutant plasmids

Label nameF1 F2 V1 V2
Template sourcePlasmid Extraction 012 pSE-EC3-ArgA
Primer (separated by semicolons)
Total volume50 μL * 4
Polymerase typeFastPfuTemplate Quantity50of
Annealing temperature65 °C/55 ℃Extension time setting70s/40s
Expected product length2.3/1·.2kb

Verify mutant plasmid amplification size (correct)

Agarose gel electrophoresis 014
Concentration0.01MarkerGenestar 2000
Voltage130 VTime25 min
Sample loading5 μL
Layout descriptionLane1: High Fidelity PCR 013F2; Lane2: High Fidelity PCR 013F1; Lane3: High Fidelity PCR 013V2; Lane4: High Fidelity PCR 013V1; Lane5: 5K Marker
DPN I template digestion 015
Label nameF1 F2 V1 V2
Total system50μL
Fixed component (automatic calculation)
10x Cutsmart buffer4.5μL
DPN I0.5μL
PCR products45μLResistance marker


DNA Fragment Recovery 016
Product NameF
Sample Source20200704 Agarose gel electrophoresis 014 Lane 1 and 2
Extraction methodTiangen universal DNA recovery kit
Elution volume30 μLDetection concentrationof / μL
RemarksMix F1 F2


Label nameG1 G2
Gibson premix10 μLType
Assemble Fragments:
DNA Fragment Recovery 0163μLNo resistance
DNA Fragment Recovery 0172μLResistance marker
Transformation 019Chemical conversion method
Tablet NameG1 7.4 HFI 、 G2 7.4 HFI
Transform DNA sources.20
DNA addition10μL
Sensitive cellDH5α Commercial Feeling State
Sensitive state usage50μL
Resuscitation mediumLB200μL
Coated plate9 cm boardLB
Resistance markerCm
Coating amountFull coating


Colony PCR and agarose gel electrophoresis to verify the product is correct


Week Three (20200706-20200712)

  • Preservation of DH5a (pSB1C3-argAfbr) and extraction of plasmid pSB1C3-argAfbr;

  • Construction of plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA fbr

Week Four (20200713-20200719)

Knockout argR gene in Nissle strain

  • Construction of plasmid pTargetA-fimA gRNA1/2-Arm-(UP&DOWN)

  • Design Information

  1. gRNA Design website:

NamegRNA sequence
  1. Primers needed to construct pT plasmids:


Week Five (20200720-20200726)

  • Preparation of Nissle competent cells

  • The plasmids pCas was transformed into competent cell

  • Inoculate pCas-Nissle, ready to make Nissle -pCas competent cell

Week Six (20200727-20200802)

  • pT was transformed into Nissle -pCas, and verify whether to knock out argR;

  • Debug:The transformation efficiency was too low,so it was necessary to reprepare Nissle -pCas competent cell.

Week Seven (20200803-20200809)

Repeat the work of the previous week: Failure

Week Eight (20200810-20200816)

  • Debug: Replace Nissle with E.coli DH10b, make competent cell;

  • The plasmids pCas was transformed into competent cell

  • Inoculate pCas-DH10b, ready to make DH10b -pCas competent cell

Week Nine (20200817-20200823)

  • pT was transformed into Nissle -pCas, and verify whether to knock out argR: succeed

  • The plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA fbr were transferred into DH10b -ΔargR

  • Culture pres

Construction of hydrogen sulfide metabolic pathway

  1. Replication of cysE gene from DH5a genome

  1. Construction of plasmids pBR322-KanR-pTac-cysE-Mut (including cysE-5, cysE-256, cysE-11-2, cysE-5-11-2,cysE-256-11-2,cysE-256-5,cysE-256-5-11-2),respecrively.

  1. Transforming pBR322-KanR-pTac-cysE-mut into Nissle competent cell.

  1. Culture preservation

Week One (20200713-20200719)

  • cysE wild-type was amplified from E.coli DH15 α genome by PCR and connected to plasmid pTYT.

  • Sequencing

Week Two (20200720-20200726)

  • Construction of plasmids pBR322-KanR-pTac-cysE-5,pBR322-KanR-pTac-cysE-256,pBR322-KanR-pTac-cysE-11-2,

pBR322-KanR-pTac- cysE-5-11-2;

  • Sequencing

Week Three(20200727-20200802)

  • Construction of plasmids pBR322-KanR-pTac-cysE-256-11-2,pBR322-KanR-pTac-cysE-256-5,pBR322-KanR-pTac-cysE-256-5-11-2;

  • Sequencing

Week Four (20200803-20200809)

  • Preparation of Nissle competent cell;

  • Plasmids pBR322-KanR-pTac-CysE-mut was transferred into Nissle competent cell, respectively.

  • After verification, the strain was preserved.

Myrcene metabolic pathway

  1. Synthesize the gene MS

  2. Construction of plasmid pBR322-KanR-pTac-RBS-GPPS-RBS-MS;

  1. Transforming plasmid pBR322-KanR-pTac-RBS-GPPS-RBS-MS into DH10b-pMevT-pMBIS competent cell;

  2. After verification, the strain was preserved.

Week One (20200810-20200816)

  • MS gene was synthesized by GenScript

  • Construction of plasmid pBR322-KanR-pTac-RBS-GPPS-RBS-MS;

Week Two (20200817-20200823)

  • Preparation of DH10b-pMevT-pMBIS competent cell;

  • After verification, the strain was preserved.

Functional testing:

Week Seven (20200824-20200830)

Test method:

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