iGEM 2019 — Team Fudan

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# Team:Fudan/Notebook

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Improve

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* [Home (current)](https://2019.igem.org/Team%3AFudan)
* Project

  <Description>
  <Design>
  <Model>
  <Safety>
  <Demonstrate>
* Experiment

  <Notebook>
  <Results>
* Parts

  <Overview>
  [Part Collection](Part_Collection)
  [Improved Parts](Improve)
  <Measurement>
* Human

  [Integrated Human Practice](Human_Practices)
  [HP Supplement](HP_Supplement)
  <Collaborations>
* Team

  <Team>
  <Attributions>

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Notebook

##### Choose a date and view our notebook:

|  |  |  |
| --- | --- | --- |
| ZDH | Resuscitative bacteria | Bacteria type：Mutaflor (Escherichia coli Nissle 1917) |
| Activation of bacteria |
| Coating plate | Begin at 13:00(BJT) in 37℃ incubator |
| Cell culture |

|  |  |  |
| --- | --- | --- |
| ZDH | Solution Configuration | LB medium (liquid)300ml×2 & (solid)300ml×2 & ddH2O×1 |
| HCZ&JKY | Solution Configuration | 0.1M CaCl2×1 & 0.1M CaCl2+15% glycerol×1 |
| In No.8 4℃ refrigerator |
| Senior Fellow Apprentice | Solution Configuration | 100mg/ml Amp×6 |
| In No.1 -20℃ refrigerator |
| HCZ&JKY | Single colony & flasks culture | Nissle×6 & DH10B×1 |
| Begin at 21:00(BJT), End at 9:00(BJT). Storage in 4℃ |

|  |  |  |
| --- | --- | --- |
| ZDH | PCR | bb1 PUC57 |
| Fail（The PCR machine clicked the Not heat cover key, and the liquid in the PCR tube was steamed dry.） |

|  |  |  |
| --- | --- | --- |
| ZDH | Receiving & preserving bacteria | DH5α×4 & W3110×2 |
| In No.8 4℃ refrigerator |
| JKY | Genome extraction | DH5α×3 & W3110×3 |
| In No.1 -80℃ refrigerator |
| Electrophoretic Verification | DH5α & W3110 (genome) |
| Image Storage "iGEM 2019 3.13" Folder. |
| PCR & Electrophoretic Verification | pUC57 backbone 1 |
| Success |
| Solution Configuration | 50mg/ml Kana×10 |
| In No.1 -20℃ refrigerator |

|  |  |  |
| --- | --- | --- |
| YYJ&WYH | PCR | (2000 and fimH two tubes each) 50 liter system |
| Inoculated-pathogen | pUC57-kan×2, Shake bacterium overnight |
| Dissolved PSB plasmid | In No.8 4℃ refrigerator |
| CYY | Competent cells preparation（Nissle） | In -80℃ refrigerator |

|  |  |  |
| --- | --- | --- |
| GYC&ZDH | Electrophoresis | fimH×2，bb-2×2，lacz×2 |
| Fail（The white shell is not compacted when mixing.） |
| ZDH | Transmission | Nissle competence was transferred to pUC57 plasmid to verify the characteristics of the covert plasmid. |
| PUC will not be lost and single colonies will grow. |
| HJY | Transmission | The amplification of DH10B into pSB3C5 failed. Preliminary analysis indicated that the quality of DH10B was too poor or that some problems occurred during the transformation process resulted in the death of competent cells. |

|  |  |  |
| --- | --- | --- |
| JKY&YYJ&HCZ | PCR | fimH×2；bb-2(Using plasmid as template); 1500&2000; lacZ×2 |
| Transmission | / |
| ZDH | Rubber Recovery | bb-2 1500&2000 pcr |
| JKY&HCZ | Transmission | bb-2 1500&2000 |
| Failure of validation may be due to primer problems or BB1 recovered before ZDH. |
| HCZ | Store lacZ parts and ptetR in Kit Plate 7 in the -20℃ refrigerator. | |
| ZDH | Flasks culture | Get DH10B from the-80℃ refrigerator |
| Inoculate three test tubes and shake them for the next day. |
| Inoculated nissle single colony shake |

|  |  |  |
| --- | --- | --- |
| YYJ | Preparation of competent bacteria | Type: DH10B |
| ZDH | Transmission | The plasmid pSB3C5 was transferred to DH10B. (It was found that it did not grow out at 3.18.) |
| ZDH&YYJ | PCR & Gel extraction | Dilution 1500 and 2000 times (bb-1). |

|  |  |  |  |
| --- | --- | --- | --- |
| ZDH | Transmission | Discarding the product recovered from yesterday's glue and re-preparing two Bb-1 tubes. | |
| The transformation failed to validate the two parts required by JKY. Preliminary analysis showed that the concentration of plasmid given by iGEM was too low. |

|  |  |  |
| --- | --- | --- |
| JKY&YYJ | Gradient PCR | pCU57-lacZ |
| PCR | pSB3C5 |

|  |  |  |
| --- | --- | --- |
| JKY | Gene Connection | lacZ and pUC57-KanR-kan pro |
| Transmission | Transform into DH10B, coating plate. |
| Fail, didn't grow, while the control group grew. |
| Electrophoretic Verification | lacZ、pUC57-KanR-kan pro、Original pUC57-KanR |

|  |  |
| --- | --- |
| ZDH | PCR of bb-2. Fail, gel image saved, approximately 1.5kb, should be around 3kb |
| ZDH&GYC | PCR of pSB3C5-vector, tet operator and fimH for one-step cloning, gel recycled |
| ZDH | One-step cloning of 3 parts mentioned above |
| LJH | One-step cloning of JKY's pUC57-kan pro |

|  |  |
| --- | --- |
| HCZ | PCR of pUC57-KanR-kan pro-BB, gel recycled |
| JKY | one-step cloning of lacZ and pUC57-KanR-kan pro-BB |
| HCZ&JKY | Transformation of recombinant product |
| ZDH&JKY | Inoculate 4 cell-culture dish, 2pUC57-KanR-kan pro-lacZ, 1 pSB3C5-CmR-fimH, 1 positive control |

|  |  |
| --- | --- |
| JKY&YYJ | PCR of lacZ from DH5α、w3110、DH10B 、fimH,gel recycled anddetecting concentrations, lacZ sequencing |
| ZDH | gel recycled, one gel image saved, pcr |
| CYY | PCR of pUC57bb,gel recycled |

|  |  |
| --- | --- |
| JKY | PCR of pGH-bb, lacZ; gel recycled; connected; transformed |

|  |  |
| --- | --- |
| JKY | Pick single colony, shake it over night |

|  |  |
| --- | --- |
| HCZ | 1）Take 8 tubes of bacteria that contain pGH-lacZ from the incubator shakers. 2) ⑤、⑥tubes are wrongly added to bacteria retaining tubes①②、③④，which waste ⑤、⑥tubes. 3)Plasmid extraction of ①②③④⑦⑧ and the protucts were in the -4℃ refrigerator. |
| JKY | pGH-lacZ was tested by HindIII, SspI, EcoRI, BglI, and the result is not satisfactory, and we were told that the backbone we use is not for expressing protein. |

|  |  |
| --- | --- |
| JKY | PCR p15A-Amp-BB and lacZ |
| DNA extraction of ZDH's 8 tube of p15A-kanR-fimH |
| YYJ | p15A-BB and lacZ from (DH5alpha) gel recycled, connected, and transformed into DH10B |
| CYY | PCR of B0015 part |
| HJY | PCR of C0061 C0062 parts, unverified. B0015 recycle complicated, remains troubleshooting. |

|  |  |
| --- | --- |
| GYC | pick 6 single colonies of DH10B P15A-lacZ, shake it overnight. |

|  |  |
| --- | --- |
| JKY | Store 2 tube of p15A-Apra-lacZ in -80℃ |
| HCZ | Miniprep of 6 tube of p15A-Amp-lacZ |
| Tested by EcoRI figure saved |
| Store 6 tube of p15A-Amp-lacZ in as well |

|  |  |
| --- | --- |
| ZDH | GFP strain incubation in 2test tube overnight |
| transformation of P15A-fimH into nissle, incubate overnight |
| Incubate DH10B with p15A-fimH in 2test tubes overnight |
| Incubate DH10B with p15A-lacZ(#2) in 2test tubes overnight |

|  |  |
| --- | --- |
| GYC | lacZ, fimH, GFP miniprep: lacZ-21.3&30.2, fimH-45.1, GFP-88 (ng/ul) |
| GFP strain cryopreserved |
| ZDH | Incubate DH10B with p15A-lacZ(#3) in 2test tubes overnight |
| Incubate nissle with p15A-fimH in 4 test tubes overnight |
| Sequencing primers of pUC-GFP sent |

|  |  |
| --- | --- |
| ZDH | Cryopreservation of Nissle-p15A-fimH-knaR strains: 14A-3-D1~8, note that DH10B-p15A-fimH-knaR strains are at 14A-3-C1~3. |
| Bacteria solution PCR of nissle-p15A-fimH-knaR to verify the existence of fimH in nissle. All lanes are bright, including negative control, suggesting nissle has fimH in its genome. |
| HCZ | Incubate cryopreserved DH10B with p15A-lacZ in 6 tubes at 9:10 am. BUT Glycerin was added by mistake! |
| JKY&YYJ | We aimed to Incubate cryopreserved DH10B with p15A-lacZ for minprep practice, but what is worth reflecting on is that we added wrong resistance(in fact it is AmpR, but we added KanR). BTW, HCZ forgot to write "AmpR" on the Bacteria retaining tube, JKY forgot to check the sanpgene before adding resistance, and YYJ forgot that once she wrote Amp on the petri dish. In a workplace, for every accident that causes a major injury, there are 29 accidents that cause minor injuries and 300 accidents that cause no injuries from |

|  |  |
| --- | --- |
| JKY | Transformation of pKD46(contains RED Homologous Recombination System) into DH10B(for amplification), incubate in 30℃ for 12-16h |
| ZDH&YYJ | Miniprep of p15A-Kana-fimH ×6 |
| JKY | Inoculation of wild Nissle(without any resistance) from bacteria retaining tube ×2, pick away at 9:00 tommorrow |
| HJY | MlacI PCR test2 succeeded |

|  |  |
| --- | --- |
| JKY | Miniprep of pKD46-Amp-30℃×4 |
| Test pKD46-Amp-30℃ by SspI |
| HCZ | Store 4 tube of pKD46-Amp-30℃ |
| JKY | Test nissle by ONPG with different propotion |
| JKY&HCZ | Transform p15A-Amp-lacZ into Nissle |
| Measure the growth curve of Nissle for 8h |

|  |  |
| --- | --- |
| JKY | Test Nissle by ONPG with different propotion again on ELIASA |
| Incubate Nissle with p15A-Amp-lacZ in 37℃ |
| YYJ | Miniprep of ZDH's pUC-GFP-AmpR-KanR ×8 |

|  |  |
| --- | --- |
| ZDH | Bacteria solution PCR of nissle-p15A-lacZ1&2, confirming plasmid to be right |
| Transmission of p15A-lacZ-AmpR into Nissle, incubate on solid LB at 37℃ |
| Incubate nissle-p15A-lacZ1&2(supposed to be contaminated) on solid LB at 37℃ overnight to purify them. |

|  |  |
| --- | --- |
| JKY | The prsentation of MRS(Anaerobic medium) |
| TPre-experiment of plate count methond(E.coli) and tube count method(anaerobion) |
| Incubate nissle-p15A-lacZ1&2(supposed to be contaminated) on solid LB at 37℃ overnight(because stupid Zhu added 150ul bacteria solution last time, and the plates were too ugly to pick a single colony) |
| YYJ | ONPG of Nissle in different lactose concentration |

|  |  |
| --- | --- |
| JKY | Presentation of LB(solid)×4 |
| Pre-experiment of plate count methond(E.coli) and tube count method(anaerobion) again with better degree of dilution |
| YYJ | ONPG for Nissle（lactose concentration0.2-2.0mg/ml）,DH10B（lactose concentration0.2-0.6） Nissle-p15A-Amp-lacZ(0.2-1.0) in different lactose concentration（bacterium concentration 20%） |
| incubate Nissle-p15A-Amp-lacZ①、② |
| pick 1 single colonies of Nissle-P15A-lacZ, shake it overnight |

|  |  |
| --- | --- |
| ZDH | Ligation of pUC-J23106-luxI-B0015-AmpR-knaR and transmitted it into DH10B, incubate in solid LB at 37℃ overnight |
| Ligation of p15A-aafA-knaR-GFP and transmitted it into DH10B, incubate in solid LB at 37℃ overnight |
| Ligation of p15A-agg3A-knaR-GFP and transmitted it into DH10B, incubate in solid LB at 37℃ overnight |
| Incubate S. cerevisiae in YPD(l) at 30℃ for 24hrs |
| HJY&CYY | OE PCR of J23106-luxI-B0015 |

|  |  |
| --- | --- |
| ZDH | Incubate p15A-aafA-knaR-GFP in liquid LB overnight (select 8 monoclone) |
| Incubate p15A-agg3A-knaR-GFP in liquid LB overnight (select 7 monoclone) |
| Incubate pUC-J23106-luxI-B0015-AmpR-KanR in liquid LB overnight (select 1 monoclone ) note that there's only one monoclone on 2plates together, I've got to make sure its not because double antibiotics were added to the petri dish. So I've put it into incubator overnight to see if new monoclones come up tomorrow morning. |

|  |  |
| --- | --- |
| ZDH | Enzyme digest of p15A-aafA-knaR-GFP and p15A-agg3A-knaR-GFP ,save fA#4&5, 3A#5, 6&8 |

|  |  |
| --- | --- |
| ZDH | Enzyme digesting fA#4&5, 3A#5, 6&8, luxI, proving only 3A#5&6 are correct |

|  |  |
| --- | --- |
| ZDH | Incubate agg3A 5-1, 6-1, DH10B, nissle, nissle-fimH in test tubes overnight |
| Select monoclone of DH10B and luxR and incubate in test tube |
| Gel recycle B0015 |
| Transform pUC-GFP-kanR-AmpR into DH10B to prove reliability of competent DH10B |

|  |  |
| --- | --- |
| ZDH | Incubate agg3A 5-1, 6-1, DH10B, nissle, nissle-fimH in Erlenmeyer. |
| Plasmid extraction of agg3A 5-1, 6-1and sent them for sequencing |

|  |  |
| --- | --- |
| CYY | PCR of LuxI, LuxR, B0015, backbone |

|  |  |
| --- | --- |
| CYY | Transmission of J23106-RBS-LuxR-B0015-bb(106) into DH10B, plated on LB agarplate with Kan and Amp Transmission of J23106-RBS-LuxR-B0015-bb(sec) into DH10B, plated on LB agarplate with Kan and Amp |

|  |  |
| --- | --- |
| CYY | CE ligation reaction of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec) Transmission of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec) into DH10B, plated on LB agarplate with Kan and Amp Failed |
| HCZ | 1)No colonies were found in the LB medium with 10-7 and 10-8 yogurt diluent after 18hours. About 10 colonies are in the MAC medium with nissle after 18hours. About 5 colonies are in the MAC medium with yogurt diluent after 18hours. 2)measuring growth curve of nissle in 3 groups for 12 hours |
| JKY | Try direct microscopic count. PCR of single colony on MAC plate, all have fimH |

|  |  |
| --- | --- |
| CYY | CE ligation reaction of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec) |
| HJY | 12 luxR-B0015 colonies miniprep，enzyme digestion test，all seems to be right |

|  |  |
| --- | --- |
| ZDH | Incubate 20ul yogurt on MRS(s)(1:100) at 37 ℃ overnight |
| Incubate 30ul nissle (cryopreserved) on LB(s)at 37℃ overnight |
| Bacteria PCR of nissle, DH10B to check the nissle-specific primer |

By HCZ

PCR, p15A-kanR-ptetR-agg3A-GFP

| Fragment | Primer Used | Tm and cycle number | Length |
| --- | --- | --- | --- |
| GFP-p15A-kanR-ptetR- | gfp-ptetR-F | 60 35cycle | 3212 |
| p15A-Vector-R |

By ZDH

Transmission to prove viability of competent cell (chemical)

| Cell name | Plasmid name | Incubate temperature | Incubate time | result |
| --- | --- | --- | --- | --- |
| DB3.1 | pUC-GFP-AmpR-kanR | 37 | 14h | Good |
| DH10B | pUC-GFP-AmpR-kanR | 37 | 14h | Good |
| Nissle | pUC-GFP-AmpR-kanR | 37 | 14h | No clone |

Inoculate bacteria in test tubes

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DB3.1 | pBR322-tetR-tetO-AmpR-cyg | Amp | 2 | 37 | 14h | Good |
|  | pBR322-tetR-tetO-AmpR-cyg | Cl | 2 | 37 | 14h | Good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result |
| --- | --- | --- | --- | --- |
| pBR322-tetR-tetO-AmpR-cyg | / | / | BcuI+Eco105I | good |
| P15A-tetR-tetO-cmR-cyg | / | / | Xbal | good |

By YYJ

CE ligation reaction of p15A-ptetR-GFP-KanR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| p15A-ptetR-GFP-KanR | p15A-ptetR-GFP-KanR | / | Good |

Transmission of p15A-ptetR-GFP-KanR into DH10B

| Cell Name | Plasmid Name | Incubate Temperature | Incubate Time | Result |
| --- | --- | --- | --- | --- |
| DH10B | p15A-ptetR-GFP-KanR | 37℃ | 14h | Good |

By CYY

PCR of luxpr-GFP, PCR of p15A backbone(AmpR)

| Fragment | Primer Used | Tm and cycle number | Length | Concentration(after gel purification) | 260/280 |
| --- | --- | --- | --- | --- | --- |
| Luxpr-GFP | Luxprfin-F | 58 35cycle | 1024 | / | / |
| Luxprfin-R | / | / | / | / |
| P15A backbone | Luxprfin-F | 58 35cycle | 1936 | / | / |
| Luxprfin-R | / | / | / | / |

CE ligation of pUC-luxI-luxR-AmpR-KanR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| pUC-luxR-AmpR | pUC-luxI-KanR | 5114 | / |

CE ligation of pUC-luxI-luxR-AmpR-KanR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| luxpr-GEP | P15A-AmpR backbone | 2920 | success |

Transmission of pUC-luxI-luxR-AmpR-KanR into DH10B

| Cell name | Plasmid name | Incubate Temperature | Incubate time | Result |
| --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR-AmpR-KanR | 37℃ | / | / |

Luxpr-E8.9
luxI:
luxR:14-2-3-d8.9
luxIR:H2.3.4
luxIR-ampR:89, purple, five tubes

By HCZ

PCR, p15A-cm-tetR-tetO-hyg-ccdB, pBR322-amp-tetR-tetO-hyg-ccdB, p15A-kanR-ptetR-agg3A-GFP

| Fragment | Primer Used | Tm and cycle number | Length |
| --- | --- | --- | --- |
| tetR-cm-p15A ori | p15A-tetR-F | 55℃ 35c | 2481 |
| p15A-tetR-R |
| tetR-amp-pBR322 ori | p15A-tetR-F | 55℃ 35c | 2688 |
| p15A-tetR-R |
| kanpro | kanpro-F | 58℃ 35c | 200 |
| kanpro-R |

CE ligation reaction of p15A-cm-kanpro-tetR, pBR322-amp-kanpro-tetR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| tetR-cm-p15A ori | Kanpro | 2481+200 | / |
| tetR-amp-pBR322 ori | Kanpro | 2688+200 | / |

Transmission of p15A-cm-kanpro-tetR into DH10B, pBR322-amp-kanpro-tetR into DH10B

| Cell name | Plasmid name | Incubate Temperature | Incubate time | Result |
| --- | --- | --- | --- | --- |
| DH10B | p15A-cm-kanpro-tetR | 37℃ | / | failed |
| DH10B | pBR322-amp-kanpro-tetR | 37℃ | / | failed |

Inoculate bacteria in test tubes

| Cell name | Plasmid name | Antibiotic concentration | Number of test tubes | Incubate Temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | p15A-kanR-ptetR-GFP | kana 10ug/ml | 5 | 37℃ | / | good |

Configuration of LB culture medium

|  |  |
| --- | --- |
| LB(s) | 2(2\*300ml) |
| Medium name | number |
| LB(l) | 1(300ml) |
| LB(l) | 1(300ml) |

By HCZ

PCR, p15A-kanR-ptetR-agg3A-GFP, pBR322-amp-tetR-tetO-hyg-ccdB, p15A-kanR-ptetR-GFP

| Fragment | Primer Used | Tm and cycle number | Length |
| --- | --- | --- | --- |
| Kanpro | kanpro-F | 58℃ 35c | 200 |
| kanpro-R |
| tetR | tetR-F | 58℃ 35c | 627 |
| tetR-R |
| backbone | bb-F | 58℃ 35c | 2996 |
| bb-R |

Send sequencing of p15A-kanR-ptetR-GFP(plasmid1), Gel Extraction

By YYJ

CE ligation reaction of p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0), Transmission of p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0）

| Cell name | Plasmid name | Incubate Temperature | Incubate time | Result |
| --- | --- | --- | --- | --- |
| DH10B | p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0) | 37℃ | 18h | / |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| P15A-kanR-AmpR-J23106-luxR-B0015 | J23106-F/R | 0.2 | / |
| P15A-kanR-ptetR-GFP-B0014 | bb-p15A-ptetR-GFP-F/R | 3.0 | / |
| pBR322-amp-tetR-tetO-hyg-ccdB | tetR-F/R | 0.7 | / |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | J23106-F/R+bb-p15A-ptetR-GFP-F/R+tetR-F/R | kanR | 37℃ | 18h | good |

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | P15A-kanR-J23106-tetR-ptetR-GFP (#1-#6) | 6 | 37℃ | 16h | good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| P15A-kanR-J23106-tetR-ptetR-GFP (#1-#6) | / | / | NdeI+XhoI | #1, #3, #5 confirmed | #1 confirmed | Cryopreserved #1, #3, #5 |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes |
| --- | --- | --- | --- | --- | --- |
| P15A-kanR-J23106-tetR-ptetR-GFP | / | / | / | good | For PCR template |

By HCZ

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23100-F+BT-R | 0.7 | 46.5 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23100-R+BT-F | 3.0 | 22.1 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23118-F+BT-R | 0.7 | 55.4 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23118-R+BT-F | 3.0 | 15.8 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23116-F+BT-R | 0.7 | 24.5 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23116-R+BT-F | 3.0 | 22.1 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23112-F+BT-R | 0.7 | 27.5 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23112-R+BT-F | 3.0 | 21.8 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23110-F+BT-R | 0.7 | 59.3 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23110-R+BT-F | 3.0 | 22.1 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23104-F+BT-R | 0.7 | 77.7 |
| P15A-kanR-J23106-tetR-ptetR-GFP | J23104-R+BT-F | 3.0 | 30.4 |

By ZDH

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | 100-F/BT-R+100-R/BT-F | kanR (1x) | 37℃ | 16h | good |
| DH10B | 104-F/BT-R+104-R/BT-F | kanR (1x) | 37℃ | 16h | good |
| DH10B | 110-F/BT-R+110-R/BT-F | kanR (1x) | 37℃ | 16h | good |
| DH10B | 112-F/BT-R+112-R/BT-F | kanR (1x) | 37℃ | 16h | good |
| DH10B | 116-F/BT-R+116-R/BT-F | kanR (1x) | 37℃ | 16h | No clone |
| DH10B | 118-F/BT-R+118-R/BT-F | kanR (1x) | 37℃ | 16h | good |

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | P15A-kanR-J23100-tetR-ptetR-GFP | 4 | 37℃ | 14h | good |
| DH10B | P15A-kanR-J23118-tetR-ptetR-GFP | 4 | 37℃ | 14h | good |
| DH10B | P15A-kanR-J23112-tetR-ptetR-GFP | 4 | 37℃ | 14h | good |
| DH10B | P15A-kanR-J23110-tetR-ptetR-GFP | 2 | 37℃ | 14h | good |
| DH10B | P15A-kanR-J23104-tetR-ptetR-GFP | 4 | 37℃ | 14h | good |
|

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| P15A-kanR-J23100-tetR-ptetR-GFP(No.1-No.4) | / | / | NdeI+XhoI | Confirmed No.2, No.3, No.4 | Cryopreserved No.2 | / |
| P15A-kanR-J23118-tetR-ptetR-GFP(No.1) | / | / | NdeI+XhoI | Confirmed | Cryopreserved | / |
| P15A-kanR-J23112-tetR-ptetR-GFP(No.1) | / | / | NdeI+XhoI | Confirmed | Cryopreserved | / |
| P15A-kanR-J23110-tetR-ptetR-GFP(No.1) | / | / | NdeI+XhoI | Confirmed | Cryopreserved | / |
| P15A-kanR-J23104-tetR-ptetR-GFP(No.1) | / | / | NdeI+XhoI | Confirmed | Cryopreserved | / |

That incubated bacteria shows green fluorescence proves there exists defects in plasmid design.. We assume that the bacterial terminator doesn't give a complete stop to tetR expressed by J23 promoters, and thus we decided to reconstruct our testing plasmid.

By ZDH

Recovery of cryopreserved bacteria

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DB10B | pSB1C3-cmR-proD-ph1F-B0015 | Cl | 2 | 37 | 14h | Good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| pSB1C3-cmR-proD-ph1F-B0015 | / | / | / | / | confirmed | As PCR template |

Recovery of cryopreserved bacteria

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DB10B | P15A-kanR-J23106-tetR-ptetR-GFP | kanR | 1 | 37 | 16h | Good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| P15A-kanR-J23106-tetR-ptetR-GFP | / | / | / | / | / | As PCR template |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| pSB1C3-cmR-proD-ph1F-B0015 | mod1-GFP-B0015-F/R | 0.2 | / |
| P15A-kanR-J23106-tetR-ptetR-GFP | mod1-GFP-F/R | 3.6 | / |

By ZDH

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | mod1-GFP-B0015-F/R+mod1-GFP-F/R | kanR(1x) | 37℃ | 18h | good |

By FSQ

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| P15A-fimH-kanR | fimH-F+OmfimH-R | 1.0 | / |
| PUCBB-GFP-AmpR | PUCBB-F+PUCBB-R | 3.367 | / |
| OmpA-linker | OmpA-linker-F+OmpA-linker-R | 0.479 | / |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | PUCBB-GFP-AmpR-kanR-fimH-ompA | Amp+kana(20+20) | 37℃ | 16h | good |

By FSQ

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | PUCBB-GFP-AmpR-kanR-fimH-ompA | 7 | 37℃ | 16h | good |

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | (mod-No.1)p15A-J23106-tetR-kanR-ptetR-GFP-B0015 #1-6 | 6 | 37℃ | 16h | good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| (mod-No.1)p15A-J23106-tetR-kanR-ptetR-GFP-B0015 #1-6 | / | / | Bacteria PCR instead | good | confirmed | Cryopreserved #1, #4, #5 |

By FSQ

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| PUCBB-GFP-AmpR-kanR-fimH-ompA(No.1-No.7) | / | / | EcoR1+Hind3 | failed | / | / |

By YYJ

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| pSB1C3-cmR-proD-ph1F-B0015 | Mod2-kanR-B0015-F/R | 0.41 | 33.7 |
| P15A-kanR-J23106-tetR-ptetR-GFP | Mod-kanR-F/R | 3.7 | 79.9 |

Recombination and transformation

| Bacteria | Plasmid | Temperature | Time | Result |
| --- | --- | --- | --- | --- |
| DH10B | P15A-J23106-tetR-B0015-ptetR-GFP-B0015 | 37℃ | 16h | good |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| (mod-No.1)p15A-J23106-tetR-kanR-ptetR-GFP-B0015 | mod2-kanR-F/R | 3.7 | 77.9 |
| pSB1C3-cmR-proD-ph1F-B0015 | mod2-kanR-B0015-F/R | 0.4 | 33.7(diluted to 3.7) |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | mod2-kanR-F/R+mod2-kanR-B0015-F/R | kanR(1x) | 37℃ | 18h | good |

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result | Notes |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | (mod-No.2)p15A-J23106-tetR-kanR-B0015-ptetR-GFP-B0015 | 6 | 37℃ | 16h | good | No green fluoresce shown |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | Sequencing | notes |
| --- | --- | --- | --- | --- | --- | --- |
| (mod-No.2)p15A-J23106-tetR-kanR-B0015-ptetR-GFP-B0015 #1-6 | / | / | PstIBacteria PCR instead | good | confirmed | Cryopreserved #4, 5 |

By FSQ

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| Nissle | fimHAD-PUC-F+fimHAD-R | 0.551 | 136.8 |
| DH10B | Ag43-fimHAD-F+Ag43-R | 2.556 | 156.1 |
| PUCBB-GFP-AmpR-kanR | Ag43-PUCBB-F+PUCBB-R | 3.367 | 98.8 |
| agg3A | agg3A-pUC-F+agg3A-R | 0.518 | 198.8 |
| DH10B | Ag43-agg3A-F+Ag43-R | 2.556 | 156.7 |
| PUCBB-GFP-AmpR-kanR | Ag43-pUCBB-F+pUCBB-R | 3.367 | 88.0 |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| (mod-No.2)p15A-J23106-tetR-kanR-B0015-ptetR-GFP-B0015 #5 | mod3-tetR-F/R | 4.0 | 55.1 |
| pSB1C3-cmR-proD-ph1F-B0015 | Mod3-tetR-b0015-F/R | 0.49 | 270.8 (diluted to 13.54) |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | mod3-tetR-F/R+Mod3-tetR-b0015-F/R | kanR(1x) | 37℃ | 18h | good |

By FSQ

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | fimHAD-PUC-F/fimHAD-R+Ag43-PUCBB-F/PUCBB-R+Ag43-fimHAD-F/Ag43-R | Amp/kanR | 37℃ | 18h | good |
| DH10B | agg3A-pUC-F/agg3A-R+Ag43-PUCBB-F/PUCBB-R+Ag43-agg3A-F/Ag43-R | Amp/kanR | 37℃ | 18h | good |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes |
| --- | --- | --- | --- | --- | --- |
| P15A-kanR-J23106-tetR-ptetR-GFP | / | / | / | good | 6 ml bacteria per EP tube |

By FSQ

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result | Notes |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | pUC-Amp/kanR-J23100-fimH-Ag43 | 6 | 37℃ | 16h | One correct | Showing green fluoresce, which shouldn’t appear |
| DH10B | pUC-Amp/kanR-J23100-agg3A-Ag43 | 6 | 37℃ | 16h | None correct | Showing green fluoresce, which shouldn’t appear |

Plan to reselect colonies. We assume its caused by not diluting template when doing PCR.

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result | Notes |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 | 6 | 37℃ | 16h | good | / |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes | Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 #1-6 | / | / | EcoRI+BglII | All good | Cryopreserved #1, 5, 6 | confirmed |

By FSQ

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result | Notes |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | pUC-Amp/kanR-J23100-fimH-Ag43 | 6 | 37℃ | 16h | good | / |
| DH10B | pUC-Amp/kanR-J23100-agg3A-Ag43 | 6 | 37℃ | 16h | good | / |

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes | Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| (pUC-Amp/kanR-J23100-agg3A-Ag43 #1-6 | / | / | BamHI+KpnI | #2, 6 correct | Cryopreserved #2, 5 | #5 correct |
| pUC-Amp/kanR-J23100-fimH-Ag43 #1-6 | / | / | XhoI | #4 correct | Cryopreserved #4 | incorrect |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 #6 | mod115-R/mod3-tetR-F | 3.34 | 133.5 (diluted to 13.35) |
| (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 #6 | mod115-F/mod3-tetR-B0015-R | 1.14 | 151.8 (diluted to 15.18) |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | mod115-R/mod3-tetR-F+mod115-F/mod3-tetR-B0015-R | kanR(1x) | 37℃ | 18h | good |

By ZDH

Select colonies and incubate

| Bacteria | Plasmid | Number of colonies selected | Temperature | Time | Result | Notes |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | (mod-115) p15A-tetR-ptetR-GFP | 6 | 37℃ | 16h | All good | / |

By CYY

CE ligation of pUC-luxI-luxR-AmpR-KanR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| PUC-luxI-Kana-Amp | LuxR | 4967 | success |

Transmission of pUC-luxI-luxR-AmpR-KanR into DH10B

| Cell name | Plasmid name | Incubate temperature | Incubate time | result |
| --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR-AmpR-KanR | 37 | / | success |

By ZDH

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes | Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| (mod-115) p15A-tetR-ptetR-GFP | / | / | EcoRI+BglII | All good | Cryopreserved #1, 2, 4, 5 | #1, 2, 4 are correct |

By CYY

Inoculate bacteria in test tubes

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR- | Amp Kana | 4 | 37 | 16h | success |

By CYY

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes | Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| pUC-luxI-luxR- | / | / | / | 1000-1, 5002-3, 5001-2 confirmed | Cryopreserved 1000-1, 5002-3, 5001-2 | 1000-1, 5002-3, 5001-2 confirmed |

By ZDH

PCR

| Template | Primer | Length(kb) | Gel recycle concentration (ng/ul) |
| --- | --- | --- | --- |
| (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 #6 | mod112-F/mod3-tetR-B0015-R | 1.14 | 240.0(diluted to 24) |
| (mod-No.3-final)p15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 #6 | mod3-tetR-F/mod112-R | 3.34 | 170.8(diluted to 17.08) |

Recombination and transformation

| Bacteria | Plasmid | Antibiotics (concentration) | Temperature | Time | Result |
| --- | --- | --- | --- | --- | --- |
| DH10B | mod112-F/mod3-tetR-B0015-R+mod3-tetR-F/mod112-R | kanR | 37℃ | 18h | / |

Standard fluoresce curve measurement.

By CYY

PCR of AmpPro-luxI-luxR-ori (PUC backbone) and Kana sequence

| Fragment | Primer Used | Tm and cycle number | Length |
| --- | --- | --- | --- |
| AmpPro-luxI-luxR-ori | NA-bb-F | 58 35cycle | 3140 |
| NA-bb-R |
| Kana | NA-KAN-F | 58 35cycle | 852 |
| NA-KAN-R |

By CYY

CE ligation of pUC-luxI-luxR-AmpR-KanR

| Fragment1 | Fragment2 | Length | Result |
| --- | --- | --- | --- |
| PUC-AmpPro-luxI-luxR-ori | Kana | 3952 | success |

Transmission of pUC-luxI-luxR-AmpR-KanR into DH10B

| Cell name | Plasmid name | Incubate temperature | Incubate time | result |
| --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR-KanR | 37 | / | success |

By CYY

Inoculate bacteria in test tubes

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR- | Kana | 6 | 37 | 16h | success |

By CYY

Plasmid extraction

| Plasmid name | Concentration | 260/280 | Enzyme digestion | Result | notes | Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| pUC-luxI-luxR-KanaR | / | / | / | #1, #2, #3, #4, #5, confirmed | Cryopreserved #1-5 | #1， confirmed |

By CYY

Transmission of plasmid into DH10B

| Cell name | Plasmid name | Incubate temperature | Incubate time | Result | Notes |
| --- | --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR-KanR | 37 | / | success | No single colony |
| DH10B | P15A-Luxpr-GFP-AmpR | 37 | / | success | No single colony |

By CYY

Transmission of plasmid into DH10B

| Cell name | Plasmid name | Incubate temperature | Incubate time | Result | Notes |
| --- | --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR-KanR | 37 | / | success | / |
| DH10B | P15A-Luxpr-GFP-AmpR | 37 | / | success | / |

By CYY

120 EP tubes of competent state: six bottles, 120 EP tubes. Inoculate bacteria in test tubes

| Cell name | Plasmid name | Antibiotics | Number of test tubes | Incubate temperature | Incubate time | Result |
| --- | --- | --- | --- | --- | --- | --- |
| DH10B | pUC-luxI-luxR- | Kana | 4 | 37 | / | / |
| DH10B | P15A-Luxpr-GFP | AmpR | 4 | 37 | / | / |

By YYJ

Plasmid extraction for template

| Plasmid name | Length |
| --- | --- |
| (mod-112-B0033)BASp15A-J23106-tetR-B0015-kanR-B0015-ptetR-GFP-B0015 | 4003bp |
| pBR322-amp-tetR-tetO-hyg-ccdB 1 | 4454bp |

By CYY

LuxI-luxR 10023-GFP KanR, AmpR G1, G2.

[click to download and view the full version of our notebook...](https://static.igem.org/mediawiki/2019/3/32/T--Fudan--Notebook.pdf)

[click to download and view the full version of our protocol...](https://static.igem.org/mediawiki/2019/0/0a/T--Fudan--Protocol.pdf)

[special protocol: acid tolerance assay](https://static.igem.org/mediawiki/2019/f/fa/T--Fudan--protocol0505.pdf)

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# Team:Fudan/Experiments

<!DOCTYPE html>

Experiments

ALTER

* [Home (current)](https://2019.igem.org/Team%3AFudan)
* Project

  <Description>
  <Design>
  <Experiments>
  <Model>
  <Safety>
  <Software>
  [Graphic Design](Graphic)
* Results

  <Results>
  <Demonstrate>
* Parts

  [New Parts](Parts)
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  [Human Practices](Human_Practices)
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* Team

  <Team>
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  <Awards>
  [Medals](Model)

Experiments

[Item 1](#item-1)

[Item 1-1](#item-1-1)
[Item 1-2](#item-1-2)
[Item 2](#item-2)
[Item 3](#item-3)

#### Item 1

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##### Item 1-1

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##### Item 1-2

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#### Item 2

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# Team:Fudan/Results

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Results

![](https://static.igem.org/mediawiki/2019/d/df/T--Fudan--resultBLACK.png) Results

[Stress resistance: hns knockout Nissle 1917](#Stress)
[Competitive capability improvement: microcin MccB17](#Competitive)
[Quorum sensing module construction](#Quorum)

#### Stress resistance: hns knockout Nissle 1917

H-NS is a transcriptional regulator which controls 5% gene expression in E. coli, including the glutamate decarboxylases GadA and GadB and the glutamate/GABA antiporter gadC. To knock out hns, we decided to use Red recombinase knock-out system (Fig. 1) and designed part BBa\_K3245003. Considering that, from our experience, Nissle 1917 is hard to conduct gene operations, we introduced approximately 500 bp length of homologous sequence flanking to provide higher knock-out affinity.

![](https://static.igem.org/mediawiki/2019/e/ee/T--Fudan--result1.png)

###### **Fig. 1 structure of part BBa\_K3245003.**

![](https://static.igem.org/mediawiki/2019/f/f3/T--Fudan--result2.png)

###### **Figure. 2 bacteria PCR testing gene hns knockout status. Primers are designed on up and down homologous sequences.** Successful knockout would yield a 1.3 kb DNA fragment and failure to knockout would yield a 2kb DNA fragment. M: λ-DNA (EcoT14 digest). +: Plasmid containing BBa\_K3245003 as template. 1-9: Different testing single colony as template.

After successfully knocking out hns in Nissle 1917, we further tested how this will affect its acid resistance ability. We conducted out experiment according to protocol listed on

![](https://static.igem.org/mediawiki/2019/6/62/T--Fudan--result3.png)

###### **Figure. 3 Acidic shock test showing the acid resistance ability of hns-knockout Nissle 1917 strains against wild type Nissle 1917.** **Top-left**: WT Nissle 1917 after acidic shock. **Top-right**: *∆hns* after acidic shock. **Bottom-left**: WT Nissle 1917 before acidic shock. **Bottom-right**: *∆hns* after acidic shock.

#### Competitive capability improvement: microcin MccB17

To enable Nissle 1917 stay in patient’s gut for longer period of time, we must improve its competitiveness against symbiotics as well as harmful bacteria. MccB17 displayed antibacterial activity on some strains of E. coli, Enterobacter, Pseudomonas and Shigella, so we chose this part to enhance the competitiveness of Nissle 1917. We ordered DNA synthesis of MccB17 gene cluster in two parts and linked it to pST-BSD plasmid, a kind of Bacterial artificial chromosome with single copy. Notably, plasmid pMccB17 is also single-copied at its nature state.

![](https://static.igem.org/mediawiki/2019/a/a7/T--Fudan--result4.png)

###### **Figure.4 construction of microcin MccB17 plasmid. oriV is a single-copy replicatior.** MccB17 (BBa\_K3245010) is divided into 2 segments and was synthesized by Generay. Co. ltd. We linked mcbA-D and McbD-G together and cloned the whole mcbA-G gene cluster into Pst-BSD plasmid. It is noteworthy that fragment pro-A consists of a natural promoter for MccB17.

![]()

![](https://static.igem.org/mediawiki/2019/0/0e/T--Fudan--result5.png)

###### **Figure.6 One of the representative disk diffusion test plates.** N, Nissle 1917 with MccB17. T1, DH10B with MccB17. K20, 20 mg/ml Kanamycin. mcH, sample bacteria that expresses MccB17 from author’s lab in which this part was first characterized.

However, all test groups show common symbiosis and faint effect of inhibiting E. coli DH10B. Due to the toxicity of MccB17, its concentration in cell is very low and it’s hard to gain precise data without purification. Further measurement is needed.

#### Quorum sensing module construction

As we have mentioned in our project description, we wanted our quorum sensing luxpR-tetR-ptetR expressing system fulfill the function of activating protection protein expression when the flora scale is small and shut off the expression of these proteins when flora scale goes high. Unfortunately, our model proved that if we stick on using wild type luxpR, our system will fail to activate ptetR as there would be too much tetR in cells even when luxpR is at its leakage state. In order to reduce the leakage intensity of luxpR so that it can initiate transcription after luxI-R dimer reaches the threshold we need, we remolded luxpR to form luxpR(HS100) and luxpR(fus100) promoter.

![](https://static.igem.org/mediawiki/2019/e/eb/T--Fudan--result6.png)

###### **Figure.7 Quorum sensing test of 4 different promoters.** Testing methods can be found in notebook-protocols. HS100: BBa\_K3245009. 10023: BBa\_K3245001. HS: BBa\_K2558001. luxpR: BBa\_R0062.

With the help of improved quorum sensing promoter, we successfully solved the problem of starting our system.

Though we have lowered the leakage of luxpR, we didn’t stop on what we had achieved. In order to further diminish the tetR level in whe whole system when luxpR is closed, we constructed single-copy plasmid, coupled with B0033, an RBS with only 1% percent the strength of B0034, to achieve minimum leakage.

![](https://static.igem.org/mediawiki/2019/2/27/T--Fudan--result7.png)

###### **Figure.8 Construction of plasmid pST-kanR-luxpR(HS100)-tetR to show an example of tetR expression system we use in our project.**

The next question that we cared about is to ensure that our system can close once luxpR is triggered. Since we were using a super-weak expression system, we would have to check whether our system could close ptetR or not. Satisfyingly, due to the ultra-strict property of ptetR and relatively high expression efficiency of luxpR(HS100), we could discover conspicuous expression strength reduction on ptetR.

![](https://static.igem.org/mediawiki/2019/9/9c/T--Fudan--result8.png)

###### **Figure.9 Figure showing activating and closing stage of luxpR-tetR-ptetR controlling system.** Left: E. coli DH10B transformed with part BBa\_K3245002, BBa\_K3245009 and BBa\_K3245013. Right: E. coli DH10B transformed with part BBa\_K3245009 and BBa\_K3245013. Both tubes were incubated at 37˚C, 220rpm overnight.

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