iGEM 2023 — Team hizju-china

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PROJECT

* [DESCRIPTION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
* [DESIGN](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
* [HARDWARE](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)
* [CONTRIBUTION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

RESULTS

* [RESULTS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
* [ENGINEERING SUCCESS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
* [PROOF OF CONCEPT](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
* [MODEL](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
* [IMPLEMENTATION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)

PARTS

* [PART COLLECTION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27part-collection%27%29%20%7D%7D)
* [PARTS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
* [PRIMER](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27primer%27%29%20%7D%7D)

LAB

* [NOTE BOOK](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
* [PROTOCOLS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)
* [SAFETY](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

HUMAN PRACTICES

* [HUMAN PRACTICES](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
* [EDUCATION AND COMMUNICATION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education-and-communication%27%29%20%7D%7D)
* [COLLABORATION](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaboration%27%29%20%7D%7D)

TEAM

* [MEMBERS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)
* [ATTRIBUTIONS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

# Notebook

![](https://static.igem.wiki/teams/4761/wiki/sub-page/cover/notebook-2.png)

# July

## July 16

### Public

Preparation of three strains with plasmid pCS-pgm-galu(kan), pSA-ARO10-adh6(cm) and
pZE-UGT85A1

## July 17

### Public

Design of primers
(pSA/pCS/pZE-KpnI-R、pZE-Xbal-F、pSA/pCS-BamHI-F、OsSJt1-Kpn1-F、OsSJt1-BamHI-R、pgm-galu-KpnI-F、pgm-galu-BamHI-R、pgm-galu-XbaI-R、aro10-adh6-KpnI-F、aro10-adh6-XbaI-R、aro10-adh6-BamHI-R)

Construction of original engineered strains using DH5α chassis.

## July 18 - 20

### Public

Amplification of engineered strains by transferring into liquid medium.

Verification of plasmid vectors and target genes using PCR and sequencing. pZE, pCS, UGT
sequencing failed.

## July 21

### Gene Knockout Part

Inoculation of pCas and pTarget strains.

## July 22

### Gene Knockout Part

Primers for knocking out (p1, p2, p3, p4 of pykA, pykF, pheA) designed.

Preservation, plasmid extraction for pCas and pTarget strains.

## July 23

### Gene Knockout Part

Transformation of pCas and pTarget plasmid into chassis, which was later transferred on
solid medium for overnight incubation.

Plasmid extraction of pCas strains, concentration evaluated.

## July 24

### Gene Knockout Part

Colonies of pCas and pTarget strains are selected and transferred into liquid medium for
amplification.

### Fermentation Part

Inoculation of pSA, pCS, pZE plasmid strains into liquid medium, incubate overnight.

## July 25

### Gene Knockout Part

Preservation, plasmid extraction, sequencing of pTarget strain. (No signals upon
sequencing)

pCas induction succeeded. Preparation of pCas plasmid competent strains. Preservation
under -80℃.

## July 26

### Fermentation Part

Inoculation of pSA, pCS, pZE strains on solid medium.

### Gene Knockout Part

Amplification of pTarget strains in liquid medium.

## July 28

### Fermentation & Gene Knockout Part

PCR verification and sequencing of pSA, pCS, pZE, and pTarget strains. pTarget induced
successfully. pSA and pCS also showed positive results.

## July 30

### Fermentation Part

Colonies of pheA knockout and pSA strains are selected for amplication. Later plasmid
extraction was made.

### Gene Knockout Part

PCR mutation, enzyme digestion for pTarget of pykA and pykF. Transformation into chassis
which was later transferred on solid medium.

## July 31

### Gene Knockout & Fermentation Part

Preparation and transformation of pheA donor.

Amplification of pSA, pZE, pCS, pTarget, pheA, pykA, pykF strains.

# August

## August 1

### Fermentation Part

Preservation, plasmid extraction, sequencing of engineered strains.

Construction of original engineered strains succeeded according to PCR verification
results. Transferred for fermentation.

## August 2

### Gene Knockout Part

Preparation and transformation of pykA donor.

Preparation and transformation of pTarget of pheA and pykF.

## August 3

### Copy Number Optimization Part

Recombination of plasmids using vectors and function genes.

### Gene Knockout Part

Transformation of pykA pTarget into pCas.

Colonies of pheA and pykF are selected and transferred into liquid medium for
amplification.

## August 4

### Gene Knockout Part

Preservation, plasmid extraction, sequencing of pheA, pykF strains.

## August 5

### Copy Number Optimization Part

Plasmid recombination failed. pZE was changed to pET. Extract original engineered plasmids(pSA, pET,
pCS)

Re-PCR verification using newly designed primers.

## August 6

### Gene Knockout Part

Re-preparation of pTarget for pykF and pheA.

Amplification of engineered plasmid strains.

## August 7

### Gene Knockout Part

Preparation of pTarget plasmids.

Attempts for recombination of plasmids.

Transformation of pykA donor.

## August 8

### Copy Number Optimization Part

Verification of recombinant plasmid strains. Positive results except pCS. Positive
plasmids are extracted and sent for sequencing.

## August 9

### Gene Knockout Part

Sequencing for pheA knockout strain succeeded. Transfer it into liquid medium for
amplification and plasmid elimination.

### Copy Number Optimization Part

PCR verification for recombinant plasmids.

## August 10

### Fermentation Part

Fermentation succeeded. Samples prepared for HPLC analysis.

### Gene Knockout Part

Extraction and sequencing of pykF pTarget.

Preservation of pheA knockout strain after liquid cultivation.

## August 11

### Fermentation Part

HPLC analysis for fermentation samples.

### Gene Knockout Part

Preparation of pCas competent strain using DH5α chassis.

Transferring pykF pTarget strain into liquid medium.

## August 12

### Fermentation Part

Fermentation showed desirable yields of rhodioloside in fermentation, but the standard
sample had problems.

### Copy Number Optimization Part

1 of 6 recombinant plasmids failed upon PCR verification.

### Gene Knockout Part

Plasmid extraction and sequencing for 5 recombinant plasmids and pykF pTarget plasmid.

PCR verification for pheA knockout strain checking its status of knockout, pTarget and
pCas induction. Electrophoresis show that pheA has been successfully knocked out.

## August 13

### Copy Number Optimization Part

Recombinant plasmids sequencing all showed negative results.

Construction of recombinant plasmids.

### Gene Knockout Part

Preservation for pheA knockout strain.

Attempt to knockout pykA gene using donor and pTarget prepared.

## August 14

### Gene Knockout Part

PCR verification again for pheA knockout strain.

Transferring pheA knockout strain on solid and liquid medium for further investigation,
according to the electrophoresis result.

## August 15

### Gene Knockout Part

Preservation and sequencing for pheA knockout strain.

PCR verification for pykA knockout strain. All pykA knockout samples turned out failed
according to electrophoresis results.

Preparation of pykF pTarget. Transformation after preparation.

## August 16

### Copy Number Optimization Part

PCR verification for recombinant plasmid strains. Strains showing positive results are
transferred to liquid medium.

### Gene Knockout Part

Amplification for pykF pTarget strain in liquid medium. Strain preserved and sent for sequencing
after amplification.

Verification for pheA knockout strain after further cultivation. All pheA knockout samples turned
out failed according to electrophoresis results.

## August 17

### Copy Number Optimization Part

All recombinant plasmid strains in liquid medium cultivated successfully, plasmids extracted and
sent for sequencing.

### Gene Knockout Part

Microscopy for pheA knockout strain. E.coli taking the major part with a little contamination by
mixed bacteria could be observed.

pykF pTarget plasmid sequencing failed. No signal.

## August 18

### Fusion Expression Part

New primers for knockout result sequencing and fusion expression plasmid synthesis designed.

Cyclization PCR for pykF pTarget plasmid construction.

### Copy Number Optimization Part

Original engineered plasmids(pSA, pET, pCS with their original functional genes) went through PCR
verification.

## August 19

### Copy Number Optimization Part

Extraction of pCS, pET, pSA plasmids, later went through concentration, enzyme digestion and
separation to obtain vectors and functional genes for plasmid recombination.

### Gene Knockout Part

PCR verification for pykF pTarget strain. Succeeded according to gel electrophoresis. Later colonies
are selected for amplification in liquid medium.

PCR verification of pheA knockout strain and pkyA knockout strain using newly designed improved
primers. pheA knockout strain showed negative results(all 3k bp bands instead of 2k) and pkyA
knockout strain showed doubtful results. pykA knockout strain was left for further investigation.

## August 20

### Gene Knockout Part

Plasmid of pykF pTarget are extracted, sent for sequencing, and its concentration was measured.

Construction of pheA knockout and pykA knockout strain by transformation.

### Copy Number Optimization Part

Verification of six vectors and genes for recombinant plasmids. Construction of recombinant plasmids
after verification succeeded. Recombinant plasmids are Transformation into chassis.

### Fusion Expression Part

Construction and purification of fusion genes adh6-linker-ARO10. (only first 2 linkers)

## August 21

### Fusion Expression Part

Construction of fusion genes adh6-ARO10. Another 4 linkers constructed and purified.

2 fusion genes made on Aug 20 collected, enzyme-processed and linked to pSA vector to form fusion
expression plasmid. Plasmids with adh6-ARO10 fusion gene are Transformation into chassis.

### Copy Number Optimization Part

PCR Verification for recombinant plasmid strains. Failed.

New plasmid vector strains(pACYCduet1, pCDFDuet1, PETDUET1, pRS-hemF) arrived. Incubate overnight.

### Gene Knockout Part

Colonies of pykF pTarget selected for amplification in liquid medium.

## August 22

### Fusion Expression Part

The rest of fusion genes (linkers 3 to 6) enzyme-processed and linked to pSA vector to form fusion
expression plasmids. Transformation into chassis.

### Copy Number Optimization Part

PCR verification for recombinant plasmid strains. Strains with positive results transferred into
liquid medium for amplification.

Plasmid extraction for new plasmid vector strains.

Primer design for new primers for verification, rPCR and Gibson Assembly® method.

### Gene Knockout Part

pykF pTarget sequencing failed. SgRNA part of its gene sequence was wrong.

## August 23

### Gene Knockout Part

Preparation of pykF pTarget plasmid using new primers and construction of pykf pTarget strain.

Preparation of pheA donor.

### Copy Number Optimization Part

Primer design for new primers for verification, rPCR and Gibson Assembly® method. Ready to send for
synthesis.

Positive recombinant plasmid strains transferred into solid medium to form colonies.

### Fusion Expression Part

PCR verification for fusion plasmid strains linker 1 and 2. Failed.

### Liposome Vesicle Part

Launched. Confirmation of protocols. Attempts for preparation of vesicles.

### RNA Thermometer Part

Launched. Confirmation of protocols. Strains with report gene EGFP ready.

## August 24

### Gene Knockout Part

Preparation of pheA and pykA donor. Construction of knockout cells by transforming corresponding
donor and pTarget into pCas competent cells.

Colonies of pykF Target transferred into liquid medium for amplification.

### Copy Number Optimization Part

PCR verification for positive recombinant strains in August 23. Failed.

Attempt of Gibson Assembly® method by building pETDuet1-ARO10-adh6 recombinant plasmid. Transferred
into DH5α chassis.

### Fusion Expression Part

PCR verification for fusion plasmid strains linker 1 to 6. Positive strains transferred into liquid
medium for amplification.

### Liposome Vesicle Part

Vesicles with desirable diameter could be observed. Later investigation in progress.

### RNA Thermometer Part

Construction of report plasmid pET-EGFP. Transferred into DH5α chassis.

## August 25

### Gene Knockout Part

pykF pTarget plasmid extracted and sent for sequencing.

### Copy Number Optimization Part

Construction of recombinant plasmid strains (vectors pET, pSA, pCS).

Attempt of Gibson Assembly® method. Failed in rPCR of the vectors.

### Fusion Expression Part

Positive strains in Aug 24 transferred into solid medium.

### Liposome Vesicle Part

Preparation of vesicles using different lecithin-cholesterol ratios. 7:4 found to be the optimal
ratio for now.

### RNA Thermometer Part

pET-EGFP strain colonies formed. Ready for verification.

## August 26

### Gene Knockout Part

PCR verification for pykA and pheA knockout strain colonies. Strains with positive results
transferred into liquid medium for amplification.

pykF pTarget sequencing failed.

### Copy Number Optimization Part

PCR verification for recombinant plasmid strains (vectors pET, pSA, pCS). Positive strains
transferred to liquid medium for amplification. Strains with doubtful results transferred to solid
medium.

Gibson Assembly® for single gene(ARO10-adh6, UGT) on three vectors(pCDFDuet, pETDuet, pACYCduet).
Six recombinant plasmids in total. Transformation into DH5α chassis and incubate overnight.

### Fusion Expression Part

PCR verification for positive strains in Aug 25. Strains with positive results transferred into
liquid medium for amplification.

## August 27

### Fermentation Part

Fermentation strains (strains with original engineered pSA, pET, pCS plasmids) transferred into
solid medium for colonies to form.

### Gene Knockout Part

pheA knockout strain transferred to solid medium.

Preparation of pykF pTarget plasmid. Transferred into chassis.

### Copy Number Optimization Part

Preservation and plasmid extraction for remaining positive recombinant plasmid strains
(pET-ARO10-adh6 and pSA-UGT). Plasmids sent for sequencing.

New plasmid strains ready for verification.

### Fusion Expression Part

Positive adh6-ARO10 fusion gene strains transferred to solid medium for further verification.

### RNA Thermometer Part

Colonies of EGFP report strains were selected and went through PCR verification. Colonies with
positive results transferred into liquid medium.

## August 28

### Fermentation Part

Colonies inoculated into liquid medium. Incubate overnight.

### Copy Number Optimization Part

PCR verification for new plasmid strains and the rest of recombinant plasmid strains with doubtful
positive results. Strains with positive results transferred to liquid medium.

Re-construction of new plasmid strains(pETDuet, pACYCduet) which showed failed in last verification
PCR.

### Fusion Expression Part

PCR verification for positive adh6-ARO10 fusion gene strains. Strains with positive results
transferred to liquid medium.

### Gene Knockout Part

pykA knockout strain transferred to solid medium.

Colonies of pykF pTarget strain transferred into liquid medium for amplification.

### RNA Thermometer Part

Plasmids of EGFP report strains extracted, sent for sequencing.

## August 29

### Fermentation Part

Strains in liquid medium transferred into flask for fermentation.

### Copy Number Optimization Part

PCR verification for the rest of recombinant plasmid strains. Failed according to gel
electrophoresis results.

Plasmid extraction and preservation for remaining recombinant plasmid strains which showed positive
results in the recent verifications. Plasmids sent for sequencing.

### Fusion Expression Part

PCR verification for the rest of adh6-ARO10 fusion gene strains. Failed according to gel
electrophoresis results.

Plasmid extraction and preservation for remaining adh6-ARO10 fusion gene strains which showed
positive results in the recent verifications. Plasmids sent for sequencing.

### Gene Knockout Part

PCR verification for pheA knockout strains. Failed.

Plasmid extraction, and preservation for pykF pTarget strains. Plasmids sent for sequencing.

### RNA Thermometer Part

Sequencing succeeded for EGFP plasmid.

Tentative design for RNA thermometer.

## August 30

### Fermentation Part

First and Second samplings.

Preparation of standard rhodioloside sample.

### Copy Number Optimization Part

Sequencing failed. Re-inoculation of strains preserved.

### Gene Knockout Part

pykF pTarget sequencing failed. Re-inoculation of strains preserved.

New strategies for gene knockout using other sgRNAs and CRISPR-transposon system. Design of new
primers.

### Fusion Expression Part

Sequencing failed. Re-inoculation of strains preserved.

## August 31

### Fermentation Part

Third sampling. Samples sent for HPLC analysis.

### Copy Number Optimization Part

Strains inoculated yesterday sent for sequencing.

PCR verification for colonies of strains pCDFDuet-ARO10-adh6 and pCDFDuet-UGT. Colonies showing
positive results were transferred into liquid medium for amplification.

### Gene Knockout Part

PCR verification for pykA knockout strain. Some colonies have completely knocked out the pykA gene
according to electrophoresis results. Transferred into liquid medium for amplification.

New pheA pTarget plasmids prepared and Transformation into chassis.

### Fusion Expression Part

Strains inoculated yesterday sent for sequencing.

### RNA Thermometer Part

RNA thermometer connected with vector pET-EGFP then Transformation into chassis.

# September

## September 1

### Gene Knockout Part

Colonies of pheA pTarget selected and transferred into liquid medium for amplification.

PCR verification for the pykA knockout strain. All showed positive results. Strains were then
transferred to another liquid medium for elimination of pTarget plasmid.

pykA knockout strain sent for sequencing.

### RNA Thermometer Part

PCR verification for colonies of RNA thermometer strains constructed yesterday. Colonies with
positive results transferred into liquid medium for amplification.

## September 2

### Fermentation Part

Reports of HPLC arrived. Further data collection in progress.

### Gene Knockout Part

pykA knockout strain transferred to solid medium for further screening of pTarget-eliminated
strains.

Extraction of pheA pTarget plasmid. Sent for sequencing.

Sequencing of pykF pTarget using bacteria solution failed. Its pTarget plasmid sent for sequencing
instead.

### Copy Number Optimization Part

Sequencing using bacteria solution failed. Revelant strains re-inoculated into solid medium.

### Fusion Expression Part

Sequencing using bacteria solution failed. Revelant strains re-inoculated into solid medium.

### RNA Thermometer Part

Extraction of pET-RNA thermometer-EGFP plasmid. Sent for sequencing.

### Gene Integration Part

Launched. Inoculation of pDonor, pEffector and pCutamp plasmid strains into liquid medium.

## September 3

### Gene Knockout Part

Screening for pTarget-eliminated pykA knockout strain.

pheA pTarget and its donor Transformation into pCas strain to build pheA knockout strain.

### Copy Number Optimization Part

Colonies inoculated yesterday were selected and went through verification PCR.

### Fusion Expression Part

Preparation of adh6-ARO10 fusion gene (with linkers 1 to 6). Ready for connection with vector pET.

### RNA Thermometer Part

Sequencing succeeded. Transformation of U6, U7 and U8 - pET plasmids into chassis to construct
reporter strains.

### Gene Integration Part

Extraction of pEffector, pDonor and pCutamp plasmids. Re-inoculation of pEffector and pCutamp
strains due to too low concentrations.

## September 4

### Gene Knockout Part

Elimination of pTarget plasmid for pykA knockout strain seems failed. Re-inoculation into liquid
medium with inducer.

Verification PCR for pykF pTarget. Mostly negative results.

### Copy Number Optimization Part

Construction of plasmids pCDFDuet-ARO10-adh6, pACYCDuet-ARO10-adh6 and pETDuet-ARO10-adh6.
Transformation into chassis.

Verification PCR for recombinant plasmid strains recently constructed. Mostly negative results.

### Fusion Expression Part

Construction of pET-(adh6-ARO10 fusion gene) plasmids with linkers 1 to 6. Transformation into
chassis.

### RNA Thermometer Part

Report strains transferred into liquid medium. Inducer IPTG added.

### Gene Integration Part

Extraction of pEffector and pCutamp plasmids.

## September 5

### Gene Knockout Part

Verification PCR for pheA knockout strain. Strains with positive results transferred into liquid
medium.

Screening for pTarget-eliminated pykA knockout strain.

### Copy Number Optimization Part

Verification PCR for recombinant plasmids(vectors pSA, pET and pCS) all failed.

Doubtful results shown during verification PCR for new vector strains(pETDuet, pCDFDuet, pETDuet).
Strains left for further investigation.

### Fusion Expression Part

Verification PCR for pET-(adh6-ARO10 fusion gene) plasmid strains inoculated yesterday. Strains with
positive results transferred into liquid medium for amplification.

### RNA Thermometer Part

Solution of EGFP report strains selected and checked for fluorescence under naked eye. No visible
phenomenon.

## September 6

### Gene Knockout Part

Verification PCR for pheA knockout strains in the liquid medium. All positive results. Transferred
into liquid medium with inducer for elimination of pTarget.

Screening for pTarget-eliminated pykA knockout strain.

### Copy Number Optimization Part

Verification PCR for new vector strains(pACYCDuet, pCDFDuet, pETDuet). Failed.

### Fusion Expression Part

Plasmids of fusion genes with positive results extracted and sent for sequencing.

Re-construction of fusion gene plasmids which failed in the last attempt. Transformation into
chassis.

### RNA Thermometer Part

Solution of EGFP report strains selected and checked for fluorescence after excitation by UV light.
No visible phenomenon.

### Gene Integration Part

Plasmids sent for sequencing.

Design of relevant primers to perform gene integration using CRISPR-transposon method.

## September 7

### Gene Knockout Part

Screening for pTarget-elimated pykA knockout and pheA knockout strains.

Construction of pykF pTarget plasmid using new primers. Transformation into chassis.

### Copy Number Optimization Part

Design of new primers for Gibson Assembly® method.

### Fusion Expression Part

Verification PCR for fusion gene plasmids constructed yesterday. Failed.

### Gene Integration Part

Construction of pDonor plasmids with ARO10 and with UGT85A1 gene. Transformation into chassis.

## September 8

### Gene Knockout Part

Screening for pTarget-elimated pykA knockout and pheA knockout strains.

Verification PCR for pykF pTarget strains. All positive. Strains transferred into liquid medium for
amplification.

### Copy Number Optimization Part

Construction of recombinant plasmids using vectors pSA, pET and pCS. Ready for biotransformation.

### Gene Integration Part

Verification PCR for constructed pDonor plasmids(both with ARO10 gene and with UGT gene). Doubtfuls
results shown. Later check for actual structure of the plasmid.

## September 9

### Gene Knockout Part

Verification PCR for pTarget-eliminated pheA knockout strain. Strains transferred into liquid medium
for amplification.

Screening for pTarget-eliminated pykA knockout strain.

### Copy Number Optimization Part

Construction of recombinant plasmid strains by biotransformation.

Construction of plasmid pCDFDuet, pACYCDuet and pETDuet linked with UGT85A1 gene. Transformed into
chassis.

### Fusion Expression Part

Fusion gene plasmids sequencing successful.

### RNA Thermometer Part

Primitive report strain(without RNA thermometer) inoculated into liquid medium. Inducer IPTG added.

## September 10

### Gene Knockout Part

Verification PCR for pTarget-eliminated pheA and pykA knockout strain. pTarget of pheA knockout strains all eliminated while pykA stayed the previous results.

pTarget-eliminated pheA knockout strain preserved and transferred into liquid medium for pCas plasmid elimination.

Continue pTarget elimination for pykA knockout strain.

### Copy Number Optimization Part

Verification PCR for recombinant plasmid strains and new vector plasmids(pETDuet, pACYCDuet, pCDFDuet with gene UGT). Strains showing positive results transferred into liquid medium.

### RNA Thermometer Part

Report strains showed green color under naked eye. Relevant strains preserved.

## September 11

### Gene Knockout Part

Screening for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain.

pykF pTarget sequencing succeeded. Ready for biotransformation for construction of its knockout strain.

### Copy Number Optimization Part

Extraction of recombinant plasmids. Sent for sequencing.

### Gene Integration Part

Preparation of pDonor plasmid using new primers. Transformed into chassis.

### RNA Thermometer Part

Inoculation of report strains U6, U7, and U8 into liquid and solid medium.

## September 12

### Gene Knockout Part

Screening for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain.

Construction of pykF knockout strain using biotransformation of donor prepared and pTarget.

### Copy Number Optimization Part

Sequencing of pACYCDuet-UGT and pETDuet-UGT plasmids successful. Ready for later assembly of ARO10-adh6 gene.

Sequencing of pCS-ARO10-adh6 failed.

### Gene Integration Part

Verification PCR for pDonor strains(with ARO10 and with UGT gene). pDonor plasmids extracted and sent for sequencing.

### RNA Thermometer Part

Verification PCR for report strains (with RNA thermometers). Strains with positive results transferred into liquid medium for amplification.

## September 13

### Gene Knockout Part

Verification PCR for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain. Gel electrophoresis result and screening result indicate that pCas plasmid of pheA knockout strain has been eliminated while pTarget of pykA knockout stayed doubuful results.

pheA strains showing positive results transferred into liquid medium without antibiotics for amplification. pykA knockout strains transferred into liquid medium with inducer and corresponding antibiotics to eliminate pTarget plasmid.

### Gene Integration Part

Plasmid extraction for pDonor plasmids.

pEffector plasmids sequencing failed. Sent for sequencing again with PCR products.

### RNA Thermometer Part

Culture medium of report strains with thermometer U6, U7, U8 transferred into well plates to observe luminescence of EGFP.

## September 14

### Gene Knockout Part

Preservation of plasmids-eliminated pheA knockout strain. Preparation of competent cells.

pykA knockout strain transferred into solid medium for later screening.

Verification PCR for pykF knockout strains. No evidence of gene knocked out. Continue cultivation to knock out the gene.

### RNA Thermometer Part

Luminescence observation failed due to wrong experiment facilities.

## September 15

### Gene Knockout Part

Verification for pheA knockout competent cells prepared yesterday. Inoculation of pheA knockout strain in case that competent cells not qualified for further biotransformation.

Verification PCR for pykF knockout strain. Failed. Retry the biotransformation.

Screening for pTarget-eliminated pykA knockout strain.

Preparation of pCas strain using biotransformation.

### Gene Integration Part

Biotransformation of pDonor-UGT and pDonor-ARO10 plasmid into chassis.

## September 16

### Gene Knockout Part

Verification of pheA knockout competent cells failed. Results indicating that cells were unable to absorb the plasmids. Colony of pheA knockout strain selected and inoculated into liquid medium.

Colony of pCas strain selected and inoculated into liquid medium.

Screening for pykA knockout strain showed that strains having pTarget eliminated appeared. Verification PCR for pykA knockout strain.

### RNA Thermometer Part

Preparation of report strains U6, U7, U8 using biotransformation.

## September 17

### Fermentation Part

Construction of engineered strains (pETDuet-ugt + pSA-aro10-adh6, pacyc-ugt + pSA-aro10-adh6, pacyc-ugt + pet-adh6-aro10(linker 3), pacyc-ugt + pet-adh6-aro10(linker 4)) prepared for later fermentation.

### Gene Knockout Part

Verification of pykF knockout strain. Failed. Biotransformation for construction of pykF knockout strain.

pTarget-eliminated pykA knockout strain transferred into solid medium for later screening for pCas elimination.

Preparation of pheA-knockout competent cells. Inoculation test for verification.

### Copy Number Optimization Part

Construction of pACYCDuet-ARO10-adh6-UGT85A1 plasmid. Transformed into chassis.

### Gene Integration Part

Verification PCR for pDonor strains to seek for successfully integrated gene. Failed.

## September 18

### Fermentation Part

pETDuet-ugt + pSA-aro10-adh6 plasmid strain failed to grow. Verification PCR for the rest of fermentation strains constructed yesterday. Strains with positive results selected and inoculated into liquid medium for amplification.

### Gene Knockout Part

Verification for pheA-knockout competent cells successful.

Screening for pCas-eliminated pykA knockout strain.

### Copy Number Optimization Part

Verification PCR for pACYCDuet-ARO10-adh6-UGT85A1 plasmid strain. Strains with positive results selected and transferred into liquid medium for amplification.

### Gene Integration Part

Verification PCR for pDonor strains to seek for existence of relevant plasmids. Successful. Results indicating that the plasmids still exist but no integration happened.

## September 19

### Fermentation Part

Fermentation strains preserved from inoculum.

### Gene Knockout Part

All pCas plasmids eliminated in pykA knockout strain. Colonies selected and transferred into liquid medium for amplification.

Verification PCR for pykF knockout strain. Failed.

### Copy Number Optimization Part

pACYCDuet-ARO10-adh6-UGT85A1 plasmid extracted from inoculum and sent for sequencing.

## September 20

### Gene Knockout Part

Preparation of pykA knockout competent cells.

### Gene Integration Part

Verification PCR for integration strains. Strains with positive results transferred into liquid medium for amplification. PCR products sent for sequencing.

## September 21

### Gene Knockout Part

Verifivation of pykA knockout competent cells failed as wrong antibiotics was used. Verification again. Preparation of new pykA knockout competent cells in case it fails.

BL21-pCas strain inoculated into liquid medium for preparation of pCas competent cells.

### Copy Number Optimization Part

pACYCDuet-ARO10-adh6-UGT85A1 sent for sequencing using new primers.

### RNA Thermometer Part

Report strains inoculated into solid medium for colonies.

### Gene Integration Part

Strains with positive results in verification PCR and sequencing transferred into liquid medium without antibodies to eliminate the tool plasmids.

## September 22

### Gene Knockout Part

Verification PCR for pykF knockout strain which failed on last trial. Positive results revealed. Strains with positive results inoculated into liquid medium for amplification.

Verification of pykA knockout competent cells successful.

Preparation of pCas competent cells canceled as all the desired genes in the project have been knocked out.

### Copy Number Optimization Part

Plasmid pACYCDuet-ARO10-adh6-UGT85A1 sequencing succeeded. Ready for biotransformation.

### RNA Thermometer Part

Colonies of report strains selected and inoculated into liquid medium for amplification.

### Gene Integration Part

Strains going through plasmid elimination inoculated into solid medium to form colonies.

## September 23

### Fermentation Part

Construction of fermentation strain pACYCDuet-ARO10-adh6-UGT85A1 by biotransformation.

### Gene Knockout Part

Preservation of pykF knockout strain and inoculation into medium with inducer IPTG for pTarget plasmid elimination.

### RNA Thermometer Part

Inoculation of report strains into 24-well plates.

### Gene Integration Part

Screening of plasmid-eliminated strains.

## September 24

### Fermentation Part

Verification PCR for fermentation strain pACYCDuet-ARO10-adh6-UGT85A1. Succeeded. Colonies selected and inoculated into liquid medium for amplification.

Construction of pheA-knockout-pSA-pET and pykA-knockout-pSA-pET strain by biotransformation. (pSA meaning pSA vector and its correspond gene ARO10-adh6, same for pET, etc.)

### Gene Knockout Part

Inoculation of pykF knockout strain into solid medium for later screening.

### RNA Thermometer Part

Concentration and fluorescence measured for report strains with time as the variant.

## September 25

### Fermentation Part

Preservation of fermentation strain pACYCDuet-ARO10-adh6-UGT85A1.

Verification PCR for pheA-knockout-pSA-pET and pykA-knockout-pSA-pET strain. Succeeded. Transferred into liquid medium for amplification.

### Gene Knockout Part

Screening for pTarget-eliminated pykF knockout strain.

### RNA Thermometer Part

Concentration and fluorscence measured for report strains at 18h.

Extraction of new report plasmid pET-28a. Construction of U6-1, U6-2, U7, U8, U9 report plasmids using this new backbone. Construction of new report strains using biotransformation.

## September 26

### Gene Knockout Part

Screening for pTarget-eliminated pykF knockout strain succeeded. Strains selected and inoculated into liquid medium for amplification.

## September 27

### Gene Knockout Part

pTarget-eliminated pykF knockout strain inoculated on solid medium to form colonies. Screening for pTarget-eliminated pykF knockout strain.

### RNA Thermometer Part

pET28a plasmid sent for sequencing.

## September 28

### Gene knockout Part

pTarget-eliminated pykF knockout strains with positive results transferred into liquid medium for amplification.

### RNA Thermometer Part

pET28a plasmids sequencing succeeded.

Verification for new report strains. Strains with positive results transferred into liquid medium.

## September 29

### Fermentation Part

Inoculation of fermentation strains on solid medium to form colonies.

### Gene Knockout Part

Preservation of final pykF knockout strain.

Section

- [July](#title-1)
- [August](#title-2)
- [September](#title-3)

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# Engineering success

![](https://static.igem.wiki/teams/4761/wiki/sub-page/cover/engineering-1.png)

# Module 1 Basic fermentation

### Cycle1

#### Design:

In order to enable E. coli engineering strains to produce salidroside, we searched for the
biosynthetic pathway of salidroside metabolism and selected one of the two metabolic pathways for
modification. L-tyrosine undergoes endogenous transamination to generate 4-hydroxyphenylpyruvate,
which is decarboxylated by the pyruvate decarboxylase gene ARO1010 (BBa\_K4761000) and then reduced
to tyrosol by endogenous ADH (BBa\_K4761001). It undergoes glycosylation by the glycosyltransferase
UGT85A1 (BBa\_K4761002) from Schizonepeta tenuifolia to produce salidroside.

#### Build:

The pSA plasmid (BBa\_K4761100) containing ARO10 gene and ADH gene, the pET plasmid (BBa\_K4761101)
containing UGT85A1 gene, and the pCS plasmid (BBa\_K4761102) containing pgm and galu genes were
directly transformed into DH5α using chemical transformation method. In the receptive state, perform
colony PCR validation, and initiate fermentation after confirming the absence of errors.

#### Test:

The results of colony PCR are as shown in Fig1.1 and verified to be correct. Through HPLC
experiments, it was found that the standard curve peak had significant fluctuations and cannot be
used as a reference.

#### Learn:

The purity of the standard substance used in this HPLC was insufficient, and after careful
literature review, it was found that the standard substance was dissolved in LB culture medium.
Afterwards, a higher purity standard of salidroside was used for the preparation of the standard
curve. In addition, no control group was set up for this fermentation.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-1.png)

Fig.1 Three plasmid colony PCR results (pSA-ARO10-adh6 validation on left 1-4, pET-UGT85A1
validation on left 5-8, and pCS-pgm-galu validation on left 9-10)

### Cycle2

#### Design:

Prepare the standard curve using a higher purity Rhodiola glycoside standard, dissolve the standard
in LB medium, and add a control group (to convert DH5α) Escherichia coli strain.

#### Build:

According to literature review, the yield of salidroside is approximately in the order of 100 mg/L,
and the standard sample is configured with a concentration of 25,50,100,200 mg/L.

#### Test:

The peak shapes of LB standard with and without salidroside are not significantly different and
cannot be analyzed.

#### Learn:

After reviewing the literature and discussing with the supervisor, it was speculated that the
components contained in LB culture medium are very complex, and some of them may be similar to the
peak time and shape of salidroside. Therefore, water and M9 culture medium will be used as solvents
for making standard curve next time.

### Cycle 3

#### Design:

Use water and M9 medium to prepare the standard solution, with a concentration gradient set to
25,50,100,200mg/L.

#### Build:

Consistent with cycle 2 operation

#### Test:

Successfully obtained the standard curve, as detailed in Fig.2 and Fig.3. Fig.2 used water as the
solvent, and Fig.3 used M9 medium as the solvent.The fermentation results of pSA-ARO10-adh6,
pET-UGT85A1 plasmid strain are shown in Fig.4, and the fermentation results of pSA-ARO10-adh6,
pET-UGT85A1, pCS-pgm-galu plasmid strain are shown in Fig.5. See the Results module for detailed
analysis.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-2.png)

Fig.2 Standard curve made with water as solvent (a: 0mg/L; b: 25mg/L; c: 50mg/L; d: 100mg/L; e:
200mg/L)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-3.png)

Fig.3 Standard curve made with M9 medium as solvent (a: 0mg/L; b: 25mg/L; c: 50mg/L; d: 100mg/L; e:
200mg/L)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-4.png)

Fig.4 Fermentation results of pSA-ARO10-adh6, pET-UGT85A1 plasmid strains (a: 24h; b: 36h)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-5.png)

Fig.5 Fermentation results of pSA-ARO10-adh6, pET-UGT85A1,pCS-pgm-galu plasmid strains (a:12h;b:
24h; c: 36h)

# Module 2 Copy number optimization

## 2.1 Triple Plasmid Skeleton Gene Matching

### Cycle1

#### Design:

To achieve an increase in the unit yield of salidroside and optimize the configuration of
metabolic pathways, we designed a strategy to regulate metabolic pathways by altering the copy
number of exogenous genes. Three types of plasmids were used: pSA (BBa\_K4761100) with low copy
number, pET (BBa\_K4761101) with high copy number, and pCS (BBa\_K4761102) with medium copy number.
We constructed six new plasmids by pairing specific genes with these plasmid skeletons. The
gene-skeleton junctions commonly utilized KpnI and BamHI cleavage sites. Our plan involved
enzymatic cleavage of the plasmid skeletons, gel recovery, and subsequent ligation to construct
the new plasmids. However, due to the small size of genes and skeleton fragments on pSA and
pCS plasmids, electrophoresis alone was not effective in separating them. To overcome this, an
additional enzyme digestion step was introduced. If the skeleton was recovered, the genes were
subjected to enzyme digestion, and vice versa. Please refer to Figure 6 for a detailed digestion
strategy.

#### Build:

use the above design to carry out the corresponding digestion. Once the digestion is completed,
proceed with gel electrophoresis to separate the fragments, followed by enzyme ligation.

#### Test:

It was found that the pSA plasmid cleaved by multiple enzymes showed no bands in the pCS plasmid
electrophoresis results, while the pET plasmid showed clear and correct size bands. The
electrophoresis results after enzyme digestion are detailed in Figure 7.

#### Learn:

Analyze the reasons and consider whether the simultaneous use of multiple endonucleases affects the
efficiency of excision, as the system using three enzymes has no bands while the system using two
enzymes has bands. Therefore, two separate enzyme digestion methods were used for pSA and pCS
plasmids.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-6.png)

Fig.6 Restrictive endonuclease selection strategy

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-7.png)

Fig.7 Electrophoresis results of three restriction endonucleases (from left to right, consistent
with Fig.6 from top to bottom)

### Cycle2

#### Design:

Two different combinations of endonucleases were used to cleave pSA and pCS plasmids, to investigate
whether the simultaneous use of multiple endonucleases affects the efficiency of each other.

#### Build:

Configure five systems as follows: Left 1, 2, and 3 with pSA plasmids and KpnI, BamHI, respectively;
KpnI and BsaI; BamHI and HindIII. Systems 3 and 4 with KpnI and BamHI of pCS plasmids; KpnI and SpeI.

#### Test:

There are still no obvious bands, as shown in Figure 8.

#### Learn:

It indicates that multiple enzymes do not affect each other's efficiency. After discussion, it was
suggested that the less conspicuous electrophoresis results may be attributed to the low copy numbers
of pSA and pCS plasmids. Because pSA is a single copy plasmid. Use the method of concentrating plasmids
to increase the concentration of plasmids, and then perform enzyme digestion operations.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-8.png)

Fig.8 Electrophoresis results after two enzyme digestion methods

### Cycle3

#### Design:

Amplify the bacterial solution from multiple test tubes, ultimately extract plasmids from 8-10
tubes, and then use a gene cleaning kit for concentration operation.

#### Build:

Concentrate according to the operation manual of the gene cleaning kit, perform enzyme digestion,
and run the gel.

#### Test:

Still no obvious bands appear.

#### Learn:

Abandon the simple enzyme digestion and enzyme ligation operation, and use PCR, enzyme digestion,
and enzyme ligation methods to reconstruct the plasmid.

### Cycle4

#### Design:

Design primers located at both ends of the gene and skeleton, paying attention to including cleavage
sites and adding protective bases at both ends of the cleavage sites. The PCR primer sequence can be
found in primer numbers 1-10.

#### Build:

Using the original three plasmids as templates, six fragments were PCR generated, including three
genes and three skeletons. Gel electrophoresis was performed to verify whether the stripe size was
correct (see Fig.9 for details). After transformation, colony PCR validation was performed to
verify that one segment of the primer was on the gene and the other segment was on the skeleton. The
specific sequence is shown in primer numbers 1-11 to 1-16, and the correct band should be 1200 (see
Fig.10 and Fig.11 for details).

#### Test:

The reconstructed plasmids pSA-UGT85A1 and pET-ARO10-adh6 were successful, while the reconstructed
plasmids pSA-pgm-galu and pCS-UGT85A1 showed significant heterobands. Further sequencing
verification failed.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-9.png)

Fig.9 Electrophoresis results of three plasmids after PCR (from left to right, 1 is the pSA
skeleton, 2 is the ARO10-adh6, 3 is the pET skeleton, 4 is the UGT85A1, 5 is the pCS skeleton,

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-10.png)

Fig.10 Reconstructed Plasmid Colony PCR Results (pSA-UGT85A1 on left 1-4, pSA-pgm-galu on left 5-7)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-11.png)

Fig.11 Reconstructed Plasmid Colony PCR Results (Left 1 is pSA-pgm-galu, Left 2-5 is pET-ARO10-adh6,
Left 6-9 is pET-pgm-galu, Left 10-13 is pCS-ARO10-adh6, Left 14-17 is pCS-UGT85A1)

## 2.2 High copy plasmid skeleton replacement

### Cycle1

#### Design:

Obtain three plasmids containing dual T7 promoter expression boxes, pACYCDuet (BBa\_K4761103),
pETDuet (BBa\_K4761104), and pCDFDuet (BBa\_K4761105). Use gibson assembly to insert the ARO10 and
adh6 genes into the first T7 expression box, and the UGT85A1 gene into the second T7 expression box.
The expression level of the second T7 expression box is relatively high. To express these three
plasmids, they need to be transferred to the BL21 (DE3) strain. Assemble two genes into the plasmid
skeleton in a step-by-step manner.

#### Build:

Use gibson assembly to integrate the ARO10-adh6 gene into the first T7 expression box, design
primers, and refer to primer numbers 1-17 to 1-26 for primer sequences. The Tm value of the
homologous sequence is 55 ℃. First, use PCR to obtain a linearized vector fragment with homologous
fragments and the gene fragment. After digestion by DpnI, perform electrophoresis gel run to verify
the correct size of the band, and then proceed with the assembly reaction.

#### Test:

The results of the electrophoretic gel run after DpnI digestion are correct (refer to Fig. 12 for
details), but the transformed validation bacteria, p results, are incorrect. The validation primers
are indicated by primer numbers 1-32 to 1-39, and there are multiple heterobands visible, as shown
in Fig. 13. The target 1000bp band is present but not very pronounced.

#### Learn:

It is speculated that the bacterial strain is impure, continue to line culture, and expect to obtain
a single colony.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-12.png)

Fig.12 Electrophoresis results after linearization of vector genes

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-13.png)

Fig.13 Colony PCR Results

### Cycle2

#### Design:

Purification of bacterial colonies with many mixed bands is expected to obtain a single band.

#### Build:

After liquid expansion culture of the colony, a single colony is drawn.

#### Test:

It was found that there were still heterozygous bands in the gel. After sequencing, the results
indicated that the genes were not integrated into the genome.

#### Learn:

Redesign primers, set the Tm value of homologous fragments to 60 degrees, and observe the results
again through experiments.

### Cycle3

#### Design:

Increasing homologous fragments may enhance assembly efficiency. Additionally, the ugt85a1 gene was
selected for integration, as the smaller fragment may result in a higher success rate of
integration.

#### Build:

Increase the Tm value of the homologous fragment from 55 ℃ to 60 ℃, with the remaining conditions
unchanged. The new primer sequence can be found in primer numbers 1-27 to 1-31.

#### Test:

Two plasmids, pacyc-UGT and pETDuet-UGT, were successfully constructed through sequencing
identification. The sequencing comparison results are shown in Fig.14 and Fig.15, and the
construction of the pCDFDuet plasmid failed.

#### Learn:

The Tm value of homologous fragments has a significant impact on integration efficiency.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-14.png)

Fig.14 Comparison of PacycDuet UGT85A1 Sequencing Results

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-15.png)

Fig.15 Comparison of pETDuet-UGT85A1 sequencing results

### Cycle4

#### Design:

After successfully integrating the UGT gene into the second expression box of the plasmid, the
ARO10-adh6 gene was integrated into the first expression box, and two pairs of primers were used
for validation in colony PCR validation to prevent the loss of the UGT gene. The primer sequences
are shown in primer numbers 1-32 to 1-39.

#### Build:

Similarly, the ARO10-adh6 gene was integrated using gibsonassembly, using a new primer designed
for loop 3 to obtain a linearized vector and gene fragments with homologous sequences.

#### Test:

Using two pairs of primers, it was found that the PCR electrophoresis bands of the colonies were all
correct, as shown in Fig.16, and the sequencing results were also correct.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-16.png)

Fig.16 Colony PCR electrophoresis verification after two Gibson reactions (left 1-8 for UGT85A1 gene
verification, right 1-8 for ARO10-adh6 gene verification)

# Module 3 Gene knockout (pheA, pykA, pykF three gene knockout)

### Cycle1

#### Design:

We plan to use CRISPR-Cas9 for gene knockout and prepare Donor fragments containing both ends of the
target gene, achieving seamless knockout through homologous recombination. Two plasmids are
involved, namely pTarget (BBa\_K4761107) and pCas plasmid containing Cas9 gene (BBa\_K4761006), which
can express sgRNA.

#### Build:

Design sgRNA using an online website (CHOPCHOP <https://chopchop.cbu.uib.no>)
and obtain pTarget plasmids containing the target sgRNA through rPCR. The primer sequences are shown
in primer numbers 2-13, 2-14, 2-19, 2-20, 2-25, 2-26. Perform further transformation and sequencing
after transformation.

#### Test:

The sequencing results showed that it was the original plasmid sequence, but the target sgRNA
sequence could not be replaced through rPCR.

#### Learn:

When using circular plasmids as templates, use DpnI enzyme digestion, otherwise false positives may
occur.

### Cycle2

#### Design:

Add DpnI digestion step after rPCR preparation of new pTarget to remove the original template.

#### Build:

Take 26ul of PCR product, add 3ul Buffer and 1ul DpnI enzyme, and react for 1.5h at 37 ℃.

#### Test:

The sequencing results are correct.

### Cycle3

#### Design:

Prepare the receptive state of bacteria containing pCas plasmid and transform the pTarget plasmid
containing target sgRNA into the receptive state containing pCas together with Donor.

#### Build:

Using the original strain as a template, Donor was prepared through one PCR and one fusion PCR. The
primer sequences are shown in primer numbers 2-1 to 2-12. The pTarget plasmid and Donor were
transferred using chemical transformation method and cultured in a 30 ℃ incubator.

#### Test:

After transformation, a large number of colonies grow on the plate, making it difficult to easily
select single colonies. In addition, there was no band during the validation of the primers, as
shown in Fig.17.

#### Learn:

A large number of colonies have grown, indicating that the concentration of pCas containing plasmid
responsive cells was too high during preparation, and the amount of transformation used was
appropriately reduced afterwards. During the validation of electrophoresis, there was a lack of
bands, and upon examination, it was found that the validation primers should be designed on the
genome.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-17.png)

Fig.17 Gene Knockout Verification Results (left side indicates not knocked out, right side has no
bands)

### Cycle4

#### Design:

Reduce the amount of pCas receptive states during conversion and redesign the validation primers.

#### Build:

Search for the location of the target knockout gene on the NCBI website and design validation
primers at approximately 1000 bp upstream and downstream, as shown in primer numbers 2-33 to 2-38.

#### Test:

It was found that there were still 3000 bands during electrophoresis verification, indicating that
the gene was not knocked out, as shown in Fig.18.

#### Learn:

The efficiency of sgRNA varies, and different sgRNAs are redesigned using online websites for
knockout experiments.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-18.png)

Fig.18 Gene Knockout Verification Results (New Primer)

### Cycle5

#### Design:

Knockout efficiency is related to the sequence of sgRNA, so multiple different sgRNA sequences are
selected to study knockout efficiency.

#### Build:

After designing the online website, the pTarget plasmid was re prepared, with primer sequences listed
in primer numbers 2-13 to 2-32 for transformation.

#### Test:

The target gene was successfully knocked out, and after comparison with the control group, the band
was verified to have changed from 3000bp to 2000bp, indicating a perfect replacement. See Fig.19,
Fig.20.

#### Learn:

We initially selected the sgRNA with the highest theoretical knockout efficiency on the website, but
the final result was that the sgRNAs ranked lower in theoretical efficiency successfully knocked out
the target gene. Therefore, when selecting relevant sgRNAs, the number of groups should be
increased.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-19.png)

Fig.19 pheA gene knockout verification (successful knockout of left 1, left 3, and left 5)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-20.png)

Fig.20 pykF gene knockout verification (successful knockout of left 1, left 3, left 6, and left 7)

# Module 4 fusion expression

### Cycle1

#### Design:

Three flexible and three rigid connectors (BBa\_K4761030-BBa\_K4761035) were designed to connect adh6
and ARO10 genes. The fusion expression essence is that the stop codon of the first gene is
replaced by the linker sequence. Since these two genes were originally on the pSA (BBa\_K4761100)
plasmid skeleton, pSA was chosen as the plasmid skeleton.

#### Build:

The two genes were amplified by primer PCR with primer numbers 3-1 to 3-14, and then the two genes
linked by linker were obtained by fusion PCR. Then the recombinant plasmid was obtained by
enzyme-cutting and enzyme-linking, and then chemically transformed into the receptive state of
Escherichia coli.

#### Test:

Use two primers 3-15 and 3-16; For the single colonies obtained by bacteria 3-15 and 1-14 p
verification, the bands obtained should be 1000 and 3000 bands, but the results were mixed (see
Fig.21), and the bands were not correct at the same time, so sequencing was carried out, and the
sequencing results were wrong, showing self-cyclization.

#### Learn:

Because the host vector used is pSA plasmid, which is single copy, the extracted plasmid
concentration is low, and the efficiency of enzyme digestion and enzyme-linking is low, other
high-copy plasmid scaffoldings are considered as vectors for fusion protein expression.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-21.png)

Fig.21 Colony PCR electrophoresis verification results (using pSA as the skeleton)

### Cycle2

#### Design:

Select the high copy plasmid skeleton pET (BBa\_K4761101) as the vector for fusion protein expression
and observe the effect.

#### Build:

After fusion expression, sequence the PCR product to determine if the linker sequence is correct.
Other experimental procedures are the same as those in cycle 1.

#### Test:

The sequencing results of the PCR product after fusion expression are partially correct. Colony PCR
validation was performed on the transformed colonies, and the bands were correct. The sequencing
results are also correct, as shown in Fig.22.

#### Learn:

When constructing a new plasmid using enzyme-linked cleavage, the concentration of the plasmid has a
significant impact on the experimental results, and it is necessary to ensure a high concentration
of the plasmid skeleton.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-22.png)

Fig.22 Comparison of pET-adh6-ARO10 Reconstructed Plasmid Sequencing

# Module 5 Gene integration

### Cycle1

#### Design:

We plan to integrate the ARO10 gene and UGT85A1 gene into Escherichia coli using CRISPR RNA guided
integrations. This system involves three plasmids: pEffector (BBa\_K4761052), pDonor (BBa\_K4761108),
and pCutamp. The pEffector plasmid contains specific sgRNA sequences that determine the location of
gene integration. The pDonor plasmid can amplify foreign gene fragments in organisms, and pCutamp
contains genes such as Cas9.

#### Build:

We integrated the ARO10 gene and UGT85A1 gene into the pDonor plasmid using the Gibson assembly
method between the Transpson Left End (BBa\_K4761050) and Transpson Right End (BBa\_K4761051).
According to the plasmid map, we designed primers to linearize the vector, with primer numbers 4-1
to 4-6. Leave a distance between the primer and two transposable elements (Transpson Left End,
Transpson Right End).

#### Test:

After linearizing the vector, electrophoretic verification was performed and it was found that the
band was around 5000, which is the first lane on the left of Fig.23. The correct value should be
around 3000, so sequencing was performed.

#### Learn:

The sequencing results showed that there was an unknown sequence between the two transposable
elements, resulting in a much larger linearized vector fragment than expected. The primers were
redesigned onto the two transposable elements to ensure that no excess fragments were inserted.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-23.png)

Fig.23 Electrophoresis results of linearized vector genes (left 1 is the pDonor plasmid skeleton,
left 2 is ARO10, and left 3 is UGT85A1)

### Cycle2

#### Design:

Using newly designed primers numbered 4-7 to 4-12, linearize the pDonor vector and two genes.

#### Build:

The experimental method is the same as cycle 1.

#### Test:

The results of pDonor linearization carrier electrophoresis are correct, as shown in Fig.24, which
is around 3000. The validation results of transformed colony PCR are correct, and the colony PCR
primer numbers are 4-13, 4-14, 4-23.

#### Learn:

Proficient in analyzing the reasons in the intermediate process, and should first test and verify
the correctness when using unknown plasmids given by others.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-24.png)

Fig.24 Electrophoresis results of the linearized vector gene with a new primer (right 1 is the
pDonor plasmid skeleton, right 2 is ARO10, and right 3 is UGT85A1)

### Cycle3

#### Design:

After the construction of the pDonor ARO10 and pDonor UGT85A1 plasmids, they were transferred into
Escherichia coli DH5α along with the plasmids pEffector and pCutamp, And after sufficient
cultivation time, we hope that the corresponding gene can be inserted into the correct position in
the genome.

#### Build:

Three plasmids were transferred into Escherichia coli using chemical transformation methods.

#### Test:

After conducting colony PCR validation, we successfully identified E. coli DH5α The three plasmids
mentioned above were transferred and the corresponding genes were successfully inserted into the
correct positions in the genome, as shown in Fig.25.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-25.png)

Fig.25 Gene Integration Electrophoresis Results

# Module 6 Liposome vesicle

### Cycle 1

#### Design:

We first plan to prepare simple liposome vesicles to verify the accessibility of liposomes and to
explore the appropriate experimental conditions for the preparation of liposome vesicles.

#### Build:

Liposome vesicles were prepared in a rotary evaporator using the thin-film rehydration method with
water for hydration.

#### Test:

At the end of the preparation, the results were characterized by dynamic light scattering and
optical microscopy, and it was found that monolayers and multilayers of liposome vesicles were
successfully prepared (showed in Fig.26).

#### Learn:

The thin film rehydration method can be a viable method for liposome vesicle preparation, although
the specific conditions may need to be further explored.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-26.png)

Fig.26 Optical microscope result of lipsome hydrated with water, PC:CH=7:2

### Cycle 2

#### Design:

After the successful preparation of liposome vesicles, we plan to prepare liposomes capable of
encapsulating E. coli.

#### Build:

The same preparation method was used as Cycle 1, but the water in hydration was replaced with LB
liquid medium containing E. coli.

#### Test:

The characterization method was the same as Cycle 1, and it turned out that in addition to monolayer
and multilayer liposome vesicles, some of the liposome vesicles could be found wrapping E. coli in
them under the optical microscope to achieve bacteria loading (showed in Fig.27).

#### Learn:

After replacing water with LB medium, the thin-film rehydration method can still successfully
prepare liposome vesicles, although some experimental conditions need to be fine-tuned (such as
rotational speed of the rotary evaporator, etc.)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-27.png)

Fig.27 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:2

### Cycle 3

#### Design:

We would like to investigate the effect of different ratios of cholesterol and phosphatidylcholine
on the formation of liposomes.

#### Build:

We set up several sets of different ratios of cholesterol and phosphatidylcholine as raw materials,
using the same preparation method as in Cycle 2.

#### Test:

The characterization method is the same as Cycle 1, and we found that the liposomes were best
prepared when the mass ratio of cholesterol and phosphatidylcholine was 7 to 2 (showed in Fig.27,
Fig.28 and Fig.29).

#### Learn:

Experiments were conducted using controlled variables to obtain the best ratio of ingredients.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-28.png)

Fig.28 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:4

![](https://static.igem.wiki/teams/4761/wiki/sub-page/engineering/figure-29.png)

Fig.29 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:6

Section

- [Module 1 Basic fermentation](#title-1)
- [Module 2 Copy number
  optimization](#title-2)
- [2.1 Triple Plasmid Skeleton Gene
  Matching](#title-3)
- [2.2 High copy plasmid skeleton
  replacement](#title-4)
- [Module 3 Gene knockout (pheA, pykA, pykF
  three gene knockout)](#title-5)
- [Module 4 fusion expression](#title-6)
- [Module 5 Gene integration](#title-7)
- [Module 6 Liposome vesicle](#title-8)

![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/teamlogo.png)

CONTACT US HiZJU\_China@outlook.com

Address: No.866, Yuhangtang Road, Xihu District, Hangzhou, P.R.China

[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/huagong-white.png)](http://che.zju.edu.cn/)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/kechuang-white.png)](https://hic.zju.edu.cn/)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/idt-white.png)](https://sg.idtdna.com/pages)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/snapgene-white.png)](https://www.snapgene.com/)

© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International
license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/hizhu-china](https://gitlab.igem.org/2023/hizju-china).

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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# Results

![](https://static.igem.wiki/teams/4761/wiki/sub-page/cover/results-2.png)

Our experiments are divided into seven parts, mainly based on molecular biology, but also involve
knowledge from other disciplines. We strive to achieve initial validation of the project design.

# Module 1 Basic fermentation

Since three plasmids were obtained from the laboratory of teacher Qipeng Yuan of Beijing University of Chemical Technology,
we planned to transform the three plasmids into DH5α receptor strains in the first module of basic
fermentation experiment. The three groups are one plasmid pSA-ARO10-adh6 and two plasmids
pSA-ARO10-adh6, pET-UGT85A1 and three plasmids pSA-ARO10-adh6, pET-UGT85A1, pCS-pgm-galu. After
transformation, the electrophoretic results of colony PCR were shown in Figure 1.1.

![](https://static.igem.wiki/teams/4761/wiki/sub-page/result/figure-1.png)

Figure1.1 Three plasmid colony PCR results (pSA-ARO1010-adh6 validation on left 1-4, pET-UGT85A1
validation on left 5-8, and pCS-pgm-galu validation on left 9-10)

After colony PCR verification was correct, we carried out fermentation experiment. Single colony was
cultured overnight before test tube, and OD value was measured to ensure that OD value was the same
as 0.05 when inoculated into shaker, and OD value was cultured to 0.3-0.4 in shaker, and inducer was
added. Samples were taken at 12h, 24h and 36h respectively. Standard sample solutions with different
concentrations were configured. We used water and M9 medium as solvents, respectively, and the
concentration gradients were set to 25,50,100,200 mg/L.

The specific data of the liquid chromatography of the standard sample solution are shown in Table
1.1, the standard curve is shown in Figure 1a, and HPLC analysis of salidroside standard is shown in
Figure 1b and Figure 1c . The detailed data of fermentation results of strains after transformation
of different plasmids are shown in Table1.1, and HPLC analysis is shown in Figure 1d and Figure 1e .

Table1.1 Detailed data of HPLC analysis

| type | Sampling time (h) | Peak time (min) | Peak area | Salidroside concentration (mg/L) |
| --- | --- | --- | --- | --- |
| Salidroside standard (Water as solvent) | 0 | 0 | 0 | 0 |
| 0 | 17.337 | 188472 | 25 |
| 0 | 17.247 | 276526 | 50 |
| 0 | 17.244 | 547456 | 100 |
| 0 | 17.243 | 1104766 | 200 |
| Salidroside standard (M9 medium as solvent) | 0 | 0 | 0 | 0 |
| 0 | 17.225 | 119006 | 25 |
| 0 | 17.236 | 311021 | 50 |
| 0 | 17.213 | 584953 | 100 |
| 0 | 17.228 | 1163694 | 200 |
| 1:pSA-ARO10-adh6 | 12 | 0 | 0 | 0 |
| 24 | 0 | 0 | 0 |
| 36 | 0 | 0 | 0 |
| 2:pSA-ARO10-adh6  pET-UGT85A1 | 12 | 0 | 0 | 0 |
| 24 | 17.613 | 32621 | 7.1891 |
| 36 | 17.601 | 28643 | 6.3935 |
| 3:pSA-ARO10-adh6  pET-UGT85A1  pCS-pgm-galu | 12 | 17.591 | 24083 | 5.4815 |
| 24 | 17.571 | 50587 | 10.7823 |
| 36 | 17.577 | 57980 | 12.2609 |

Table 1.1 Detailed data of HPLC analysis (Contains standard products and samples)

![](https://static.igem.wiki/teams/4761/wiki/sub-page/result/figure-2.png)

Section

- [Module 1 Basic fermentation](#title-1)
- [Module 2 Copy Number Optimization Module](#title-2)
- [2.1 Original Vector Swap](#title-3)
- [2.2 Dual Expression Vector Installation](#title-4)
- [Module 3 Gene knockout module](#title-5)
- [Module 4 Fusion express](#title-6)
- [Module 5 Gene Integration](#title-7)
- [Module 6 RNA thermometers](#title-8)
- [Module 7 Lipsome vesicle](#title-9)

![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/teamlogo.png)

CONTACT US HiZJU\_China@outlook.com

Address: No.866, Yuhangtang Road, Xihu District, Hangzhou, P.R.China

[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/huagong-white.png)](http://che.zju.edu.cn/)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/kechuang-white.png)](https://hic.zju.edu.cn/)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/idt-white.png)](https://sg.idtdna.com/pages)
[![](https://static.igem.wiki/teams/4761/wiki/home-page/logo/snapgene-white.png)](https://www.snapgene.com/)

© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International
license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/hizhu-china](https://gitlab.igem.org/2023/hizju-china).