iGEM 2017 — Team TJU_China

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# Team:TJU China/Notebook

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CHINA](https://2017.igem.org/Team%3ATJU_China)

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## Notebook

#### [April](#April)

We determined the main project to be the new applications of the novel fluorescent protein smURFP. We came up with 3 directions one after another to rich our project. The first is to reform the protein structure. The second is to detect BV in bird’s blood. The third is to make intestinal bacteria tracking in vivo in real time come true. To achieve these goals, we thought deeply for best arrangement. For structure and BV detection, we need purified protein and BV. For intestinal tracking, we choose surface display and co-expression for different kinds of bacteria. For solving the problem of the membrane permeability of biliverdin, we selected the surface display the target protein with the biliverdin connected outside the bacteria in anaerobes or co-express the smURFP and the enzyme, HO-1, to produce the biliverdin inside the bacteria. We first chose 7 of the bacteria, which is of vital significant in human intestine, as our research subject.

#### [May](#May)

#### 1stweek

##### Detection BV and the Structure of smURFP

We read the paper of the targeted protein, smURFP, of Mr.Qian carefully, and exchanged the amino acid sequence for prokaryotic expression to get better effect of expression.

##### EHEC

We got the plasmids of PACYC184 and received the strain we need from YiGang Xu teacher

We designed the primers about surface display and sent to the company to synthesize

##### B. fragilis

We found a lot of paper and finally found a paper on the Cell magazine of a research team from Stanford University who did a similar work as we are going to do. Then our group had several deep discussion about that paper and decided to send an e-mail to Dr. Liz Stanely asking for the sharing of plasmid pWW3534, pWW3515, pWW3452, pWW3535, pWW3536 and the original pNBU2, also with some questions about the RBS they use in their designs. Besides, we studied the technique of molecular cloning in the lab.

##### L. Iactis

Enter the laboratory to learn the basic experimental skills of preparation for solid media and molecular cloning.

Design primers to align smURFP, RBS and HO-1 genes.

##### C. rodentium

We searched the reference to get proper plasmid. And then we found that we could use Pacyc184, the same with EHEC. In addition, we chose two promoter for alter. We also sent email for the bacterial strain but failed.

##### B. longum

We found a lot of material and paper, selected the anchoring sequences for bifidobacteria surface display, selected several promoters as backup, and developed a protocol for the construction of shuttle plasmids. Nevertheless we studied the technique of molecular cloning in the laboratory.

#### 2ndweek

##### Detection BV and the Structure of smURFP

We got new complex gene of smURFP for prokaryotic expression, and of course, tried to pick up the gene to construct the plasmid with smURFP.

##### EHEC

We extract the plasmid from the glycerin bacterias of last year

##### B. fragilis

We got a reply from Dr. Liz Sranely of his promise to send us the plasmids. We designed primers for smURFP and HO-1, and also for the five anchoring sequences-INPN, INPNC, LPP-OMPA, Brka and AIDA. At the same time we continue to study in the lab and mainly the PCR.

##### L. Iactis

Obtain the expression vector pMG36e, L. Lactis strain NZ9000 and the culture method with the generous help of Nankai University

##### B. longum

We continued to study in the lab and actively promote our own research. We transformed the two plasmids from Jilin University into Escherichia coli. Three clones were picked and cultured respectively, and the plasmids were extracted successfully.

#### 3rdweek

##### Detection BV and the Structure of smURFP

We successfully constructed the plasmid of PET-28b (+) with smURFP and transformed it to the bacteria for expression, E.coli BL21.

##### EHEC

We started the first round of PCR to get the target fragments of four kinds of surface display; and then continued the second round of PCR to connect two fragments. We succeeded in completing the connections of some fragments, but some failed. Now some surface display is undergoing the third round of PCR and the others are still continue the second round.

##### B. fragilis

We activated five types of E.coli with our anchoring sequences in their plasmids. Then the plasmids with anchoring sequences are extracted respectively and are ready for doing the first round of PCR experiment. Also, we add His tag in our primer and found proper restriction enzyme cutting sites according to the paper.

##### L. Iactis

2 rounds of Overlap PCR to obtain our target gene.

Activate E. coli containing pMG36e and extract the plasmid after amplification.

Transform shuttle plasmid pET28bs into DH5α, and extract the plasmid.

##### B. longum

Because we cannot obtain the synthesized anchoring sequence, we modified the entire protocol and designed the corresponding primers. At the same time we have selected the appropriate kit for extracting the genome of the strain.

#### 4thweek

##### Detection BV and the Structure of smURFP

We tested the inducing conditions and the conditions of purification to get more yield of smURFP. And luckily, we got the purified smURFP.

##### EHEC

We started the first round of PCR to get the target fragments of four kinds of surface display; and then continued the second round of PCR to connect two fragments. We succeeded in completing the connections of some fragments, but some failed. Now some surface display is undergoing the third round of PCR and the others are still continue the second round.

##### B. fragilis

We activated five types of E.coli with our anchoring sequences in their plasmids. Then the plasmids with anchoring sequences are extracted respectively and are ready for doing the first round of PCR experiment. Also, we add His tag in our primer and found proper restriction enzyme cutting sites according to the paper.

##### L. Iactis

2 rounds of Overlap PCR to obtain our target gene.

Activate E. coli containing pMG36e and extract the plasmid after amplification.

Transform shuttle plasmid pET28bs into DH5α, and extract the plasmid.

##### B. longum

Because we cannot obtain the synthesized anchoring sequence, we modified the entire protocol and designed the corresponding primers. At the same time we have selected the appropriate kit for extracting the genome of the strain.

#### [June](#June)

Preparing for the final examinations.

#### [July](#July)

#### 1stweek

A week off. A few members are preparing for some reagents.

#### 2ndweek

##### Detection BV and the Structure of smURFP

We tested several conditions for smURFP to get the crystal of smURFP.

##### EHEC

Measuring biomass of EHEC completed with a growth curve.

Competent cells of DH5α prepared.

Extracting standard plasmids.

Failing of constructing standard modules of LPP, INPNC, INPN, AIDA.

##### B. fragilis

First round PCR for AIDA, INPN, INPNC and LPP.

##### C. rodentium

We finished overlap PCR of co-expression and INPNC. And we dealt with the freeze-dried powder of Citrobacter rodentium following the direction while getting some infectious microbe in the access of culturing.

##### B. longum

Try to overlap GLBP with HU promoter. But it is hard to do that, so we failed several times.hree clones were picked and cultured respectively, and the plasmids were extracted successfully.

#### 3rdweek

##### Detection BV and the Structure of smURFP

We sent the crystal to Shanghai for the information of the diffraction points of it. And then, Chen Wu started the work of analyzing the structure of smURFP.

##### EHEC

Cultivation of bacteria stored in glycerol and failing to extract plasmid pACYC184.

##### B. fragilis

Second and third round PCR for AIDA, INPN, INPNC and LPP.

##### L. Iactis

Amplify the Smurfp、HO-1 and linker（the RBS sequence from the PMG36e and papers①）

Overlap PCR get the Smurfp-linker- HO-1

Activated Glycerol DH5α that contain PMG36e

Pick the monoclonal colony and purify plasmid, however, the result of the agarose gel electrophoresis is blank.

##### C. rodentium

We finished overlap PCR of Brka. And we fall in the DH5α conversion of coexpression-Pet28a plasmid and INPNC--Pet28a plasmid.

##### B. longum

We changed the strategy which separate GLBP into two sediments to make overlap more easier. However it was a pity that PFU protease which produced by our own lab cannot do PCR very well with the goal sequence is over 2000bp. So the property of the production was bad for further experiments.

#### 4thweek

##### EHEC

Linkage of lpp, INPNC and INPN to vector pET28-a confirmed.

Making an appointment of usage of confocal.

##### B. fragilis

Enzyme dygestion and lignation of the surface display construction.

Make Competent cell of E.coli DH5α λ-pir for amplification of plasimd pWW3536.

##### L. Iactis

Get the new Glycerol DH5α that contain PMG36e and repeat the same experiment as last week

Digest the PMG36e and Smurfp-linker- HO-1 with Xba I and Hind III,ligate them together

Transform them into DH5α，but the result was not as we expected.

##### C. rodentium

We tried to Double Digest and convert of coexpression-Pet28a plasmid, INPNC--Pet28a plasmid and Brka-Pet28a plasmid and Brka-Pet28a plasmid was finished.

The Citrobacter rodentium was identified and isolated from the mixed strain using EMB culture medium and subcultured.

##### B. longum

This week we modified the previous plan. After failing for two times, we decided to try a new method called seamless cloning which main advantage is no need of digestive sites, thus it can definitely help us save a lot of time. We made two programs about constructing surface display simultaneously: one is to connect the HU-meaningless sequence-smURFP and link them into the pGH-PMB1 vector, after finishing this transformation, we finally insert the GLBP sequence into the constructed vector to replace the meaningless sequence. The other option is to connect the three segments of HU, GLBP, smURFP with overlapping regions and directly into the PGH-PMB1 vector.

In the end, we succeeded in connecting the HU-meaningless-smURFP to the PGH-PMB1. In addition, we successfully construct the co-expression vector.

#### [August](#August)

#### 1stweek

##### EHEC

Co-expression confirmed via confocal.

Extracting plasmid pacyc184.

Extracting plasmid pacyc184.

##### B. fragilis

Repeat doing Competent cells.

##### L. Iactis

Prepare the new Erythromycin solution（50mg/ml）、 LB solid medium that contain Erythromycin（200mg/ml、300mg/ml）

Repeat the same experiment as last week，there are some special monoclonal colony，but the purify plasmids are also blank by agarose gel electrophoresis.

##### C. rodentium

Co-expression and INPNC were reconnected and converted and INPNC-Pet28a plasmid was finished. We tried to convert the DNA fragments to the pACYC184 plasmid.

##### B. longum

We finished our experiments about the construction of the plasmids. The co-expression vector and the surface display vector which GLBP as the anchoring sequence has been sequenced and the results are positive.

#### 2ndweek

##### EHEC

Ligation product of co-expression and INPN to plasmid pacyc184 available. Sequencing analysis is processing.

##### B. fragilis

Considering that we may have little possibility to do this bacterium well, we regretfully suspended the plan.

##### L. Iactis

Considering that we may have little possibility to do this bacterium well, we regretfully suspended the plan.

##### C. rodentium

We tried to make up the competence of Citrobacter rodentium and succeeded. And we finished the INPNC-Pacyc184 plasmid.

##### B. longum

We read some references and learned that a kind of anchoring sequence called ACMA could be widely used in the Gram positive bacteria, so we decided to have a try whether it could function or not. And we successfully constructed the plasmid for ACMA.

Then we try to do electro transformation for the first time. But we cannot sure whether it was succeeded or not since after we extract the plasmids and do gel electrophoresis, nothing was visible on the gel.

#### 3rdweek

##### Detection BV and the Structure of smURFP

We tried to product the protein, smURFP, and purified it from E.coli BL21. The first time, we tested whether smURFP can combine with Biliverdin (BV) and emit infrared fluorescence.

##### B. fragilis

Try to do electro transformation and verification for the second time. But this time there were no monoclone on the plate.

#### 4thweek

##### Detection BV and the Structure of smURFP

We setted a seires of ratio of smURFP and BV to test which condition is the best for emit infrared fluorescence.

##### EHEC

Preparing competent cells of EHEC.

Transforming vectors to EHEC failed.

Construction of standard modules.

##### C. rodentium

We failed in the coexpression-Pacyc184 plasmid and Brka-Pacyc184 plasmid.

We converted INPNC-Pacyc184 plasmid into the competence of Citrobacter rodentium, while see two kinds of bacterial colonies. So we used EMB culture medium to identified and isolated the Citrobacter rodentium.

##### B. longum

Try to do electro transformation and verification for the third time. This time we get the monoclone but we faced the problem we faced in the first time. We suspected whether the plasmid was into the bacteria or not. So we search the reference and modified the procedure.

#### [September](#September)

#### 1stweek

##### Detection BV and the Structure of smURFP

We collected the data of the test of the ratio of smURFP and BV, plot the relation schema of them and drew a conclusion.

##### EHEC

Regaining competent cells of EHEC.

Construction of standard modules.

##### C. rodentium

We finished the coexpression-Pacyc184 plasmid. And we converted INPNC-Pacyc184 plasmid into the competence of Citrobacter rodentium one more time and picked the monoclonal, made plate lithography, and extracted the plasmid to verify.

##### B. longum

Try to do electro transformation and verification for the fourth time. We realize that the ORI of B. longum had a low copy number, so it is not difficult to image why we always failed in extracting plasmid with 5 ml bacteria. Then we expand the culture quantity for more plasmid. But we still had difficulty in verification with enzyme digestion since the concentration were still not high enough.

#### 2ndweek

##### EHEC

Testing fluorescence using Microplate Reader for E.coli BL21 of co-expression system.

Construction of standard modules.

##### C. rodentium

Changeing P2 on to the plasmid.

Construction of standard modules.

##### B. longum

Plasmid extraction for verification.

Construction of standard modules.

#### 3rdweek

##### EHEC

Using confocal microscopy for testing fluorescence of E.coli BL21.

Construction of standard modules.

##### C. rodentium

Plasmid extraction for verification (with new promoter P2).

Construction of standard modules.

##### B. longum

Construction of standard modules.

#### 4thweek

##### EHEC

Try to transform plasmid into new competent cells of ATCC35150.

Construction of standard modules.

##### C. rodentium

Plasmid extraction for verification (with new promoter P2).

Construction of standard modules.

##### B. longum

Construction of standard modules.

#### [October](#October)

#### 1stweek

The National Day！

#### 2ndweek

##### EHEC

Regaining competent cells of EHEC.

Doing in vivo experiments on mice in Nankai University using engineered E.coli BL21.

Construction of standard modules.

##### C. rodentium

Try to do western blot. But nothing is on the gel.

Construction of standard modules.

##### B. longum

Construction of standard modules.

#### 3rdweek

##### EHEC

Doing western blot of E.coli BL21 with plasmid carried INPNC suface display construction.

Construction of standard modules.

##### C. rodentium

Construction of standard modules.

##### B. longum

Construction of standard modules.

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# Team:TJU China/Experiments

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## Experiments

### Molecular Cloning

### Take smURFP standardization for instance

#### Transfer

1. Mix the smURFP Plasmid (pET 21-b, Amp+, 1μL 100ng/μL) with the E.coli DH5α competent cells in the EP tubes. Incubate them on ice for 20-30 minutes. \* Plasmid pET21-b plasmid containing the Amp-resistant gene of smURFP was selected.

  2. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 90 seconds without shake. Transport the tubes in ice for 3-5 minutes.

  3. Add 550 μL of LB media to each transformation. Incubate the cells at 37ºC, 220RPM for 30 minutes while the tubes are shaking to express the Amp resistant gene.

  4. Amp-resistant plates were preheated in a 37℃ incubator.

  5. Put the tube centrifugal 2 min, 3000 RPM. Remove 500μl the supernatant.

  6. Plate 100 µl of the transformation onto the dishes with LB agar (AMP), and spread.

  7. Resuspend E. coli pellets. Incubate the plates at 37ºC for 12-25 hours, making sure the agar side of the plate is up.

#### Pick Monoclonal colonies and transferred to ampicillin-resistant LB medium for subculture

In the clean bench, a single colony was picked up from the medium with a pipette tip, and introduced into a test tube containing 5 mL of LB broth and 5 μL Amp, covered with a stopper, shaken at 220 rpm at 37 ° C Bed culture 12 ~ 16h.

#### Extract the amplified plasmid from the cultured Escherichia coli

1. Add the E. coli solution into the EP tube

  2. Re-suspend pelleted bacterial cells in Buffer P1 BL (kept at 4 °C) and transfer to a micro-centrifuge tube.

  3. Add Buffer P2 and gently invert the tube 4–6 times to mix.

  4. Add 350μl Buffer N3 and invert the tube immediately and gently 4–6 times.

  5. Centrifuge for 10 min at 12,000 rpm in a micro-centrifuge.

  6. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm after static for 2min. discard the flow-through.

  7. Add supernatant from the EP tube to the column and put it into collection canals. Add 500μl Buffer BL and centrifuging for 1min at 12000rpm. Discard the flow-through. Spin for 60 seconds produces good results.

  8. Adding 600μl Buffer PW and centrifuging for 60s after static for 2min. Discard the flow-through.

  9. Repeat step 8.

  10. Centrifuging for 2min at 12000rpm to shake off the rest of the Buffer PW.

  11. Place the column in a new EP tube and the opening was allowed to stand for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.

  12. Add 50μL 75℃ of sterile distilled water at 75 ° C drop wise to the middle of the adsorbed film. Static for 2min.

  Discard the flow-through. Centrifuging for 2min at 12000rpm.

#### Examine the concentration of the extracted plasmid

Add 1 drop of sterile distilled water at the light hole of nano Drop and erase it after 90s for two times. Add 1 drop of the solution in EP tube at the light hole of nano Drop with pipette and erase it after measure.

#### PCR

We use overlap PCR to get all the fusion protein. To splice two DNA molecules, special primers are used at the ends that are to be joined. In first cycle we use a couple of primers of and the same to B to PCR. By this, we design a same length of base sequence. In second cycle, due to the homologous sequence, when adding the beginning primers and the final primers, A and B can be primers to each other. They extend to a combined fragment.

#### Agarose Gel Electrophoresis(Plasmid & PCR product) ：

1. Prepare sufficient lx TAE to fill the electrophoresis tank and to cast the gel.

  2. Prepare a solution of agarose in electrophoresis buffer at a concentration of 1%: Add 0.9g powdered agarose to 90ml of TAE in an Erlenmeyer flask.

  3. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.

  4. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 μg/ml. Mix the gel solution thoroughly by gentle swirling.

  5. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.

  6. Pour the warm agarose solution into the mold.

  7. Allow the gel to set completely (30-45 minutes at room temperature), then carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape Mount the gel in the electrophoresis tank.

  8. Add just enough electrophoresis buffer to cover the gel to a depth of ~1 mm.

  9. Mix the samples of DNA with 10 μl green buffer

  10. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micropipettor. Load size standards into slots on both the right and left sides of the gel.

  11. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.

  12. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.

#### Plastic recycling

1. Column regeneration: add 500μl balance liquid to adsorption column CA2 12000 rpm centrifugal 1 minutes, pour out the filtrate and put it back to the collecting canals.

  2. Cut the DNA band from the gal, weighing in the clean centrifuge tube and weight.

  3. Add equal volume solution PN (such as 0.1 g gal, to join 100μl PN) in rubber block, then put in 50 ℃ water bath place, turning up and down continuously in order to ensure fully dissolved. Cooling to ambient temperature.

  4. Add the solution into an adsorption column CA2 under the ambient temperature for 2 minutes. 12000 rpm centrifugal 30-60 seconds, pour out the filtrate, placed at ambient temperature for a few minutes to ewnsure fully getting rid of the alcohol. Put the adsorption column CA2 into the collection canals.

  5. Drift lotion to column to join 600 μl PW, 12000 rpm centrifugal 1 minutes, pour out the filtrate.

  6. Repeat step 5.

  7. 12000 rpm centrifugal for 2 minutes, pour out the filtrate, placed at ambient temperature for a few minutes to ensure fully getting rid of the filtrate.

  8. Put CA2 in clean centrifuge tube, add ddH2O and wait for 10 minutes at ambient temperature, 12000 RPM, centrifugal 2 min. collect the DNA solution.

  Saved the DNA concentration at -20 degrees Celsius to prevent DNA cleavage.

#### Digestion of PCR products

Add 1μL DMT enzyme, PCR products, buffer and water to 20 µL, mix, 37 ℃ water bath heating 1h.

#### Ligation

Mix 1 µL T4 DNA Ligase, 2 µL ligase buffer, 50ng Vector Plasmid, Insert DNA (molar amount is 5 more than plasmid.) and sterile water (Make reaction up to 20 µL). Then incubate at25℃ for 30 - 60 min or at 16℃ for 60min.

#### Electro-transformation (B. longum)

1. Incubate bacteria anaerobically in MRSS broth (+0.5M sucrose) over night.

  2. Transfer culture to 50ml MRSS broth and incubated at 37°C until the culture reached OD600 of 0.5 to 0.7.

  3. The cells washed third times in ice-cold sucrose-citrate buffer (0.5 M sucrose, 1 mM ammonium citrate [pH 6.0]).

  4. Gather the cells in 200 μl of the same buffer.

  5. Electroporation was performed at 25 μF, 200 Ω, 12.5 kV (0.2mm) and 4ms for 100μl cells and 0.6ng plasmid.

  6. The cells were immediately diluted in 900 μl of MRS broth and incubated for 2.5 h-3h.

  7. Spread the cells onto Amp+ MRS agar plate.

  8. Plates were incubated for 2 to 3 days at 37°C under anaerobic conditions.

### Structure Analysis of Protein

#### A large number of cultured protein expression bacteria

1. Extract 5μL of the bacterial strain from the bacterial strain retained in the test expression, and add 5μL Amp to a test tube containing 5 mL of LB culture solution.

  2. Incubate the plates at 37 ° C overnight at 220 rpm in a shaker

  3. Pour the bacteria in the test tube into a 2L Erlenmeyer flask containing 1 L of LB medium, and add 500μL Amp.

  4. 37 ℃, 220rpm in the shaker culture 4 ~ 5h, on behalf of bacteria growth to logarithmic phase (OD value 0.6-0.8) 16 ℃, 220rpm temperature drop 1h (temperature dropped to 16 degrees), and then add 500μL IPTG induced E. coli (4) Producing the protein of interest.

  5. 16 ℃, 220rpm overnight culture 12h ~ 15h.

  6. Collection: The bacteria were centrifuged at 4000rpm for 16min at 4000rpm and the supernatant was discarded. The precipitated Escherichia coli was transferred to a 50mL centrifuge tube and stored at -30 ℃ (total of 6 vials)

#### Purification of SmURFP

1. Disrupt the bacteria by high-pressure sterilizers, and the broken bacteria were ultrasonographed for 15 minutes (4S ultrasound, 6S interval) to remove the nucleic acids carried by the protein of interest. Followed by equilibration in a high speed centrifuge tube, 18000 rpm for 20 minutes, separating the supernatant containing the protein and the precipitate containing the cellular debris.

  2. Nickel column：The supernatant was applied to the His-Accept nickel column. After washing unbound proteins with the lysis buffer (50mM Tris-HCl, pH 7.5, 300mM NaCl, 20mM imidazole), the bound proteins were eluted with elution buffer (50mM Tris-HCl, pH 7.5, 300mM NaCl, 250mM imidazole)

  3. A / B Formulation A: pH 5.0 50mM Sodium Acetate 50mM NaCl B: pH 5.0 50mM Sodium Acetate 1 M NaCl

  4. Cation exchange column: The sample eluted from the nickel column was added to a 10kd concentrating tube. The solution was concentrated at 3500 rpm to a solution of cation exchange column A, diluted ten times with A solution to 5mL. And then separated and purified by Hitrap SP HP 5mL column to obtain a peak shape. According to the shape of the peak were collected in the collection tube sample run glue.

  5. Molecular Sieve: Samples obtained from the S-column were changed (75% solution A + 15% solution B) and concentrated to 500μL. Using AKTA system, the superdex75 molecular sieves with appropriate separation range but higher resolution were used to purify the target protein. Therefore, the corresponding protein samples were collected. Concentrated and re-separated through molecular sieves, respectively. Respectively,
6. Crystallization: Take the corresponding position of the sample, measured with NanoDrop its concentration. According to the residual amount of protein, we used the conditions to do local screening of samples.

### Standard Curves of BV, smURFP and RFU

1. Get the pure smURFP as shown above and dissolve in protein solution.

  2. Dissolve BV in DMSO for stock, keep in the dark place.

  3. Add different amount of protein solution and BV stock solution in black 96-well plate in the dark.

  4. Use protein solution for the rest volume (up to 50μl).

  5. Incubate for 30 min.

  6. Use micro plate reader for RFU.

  7. Use Origin for standard curves.

### Protein Expression in Host Bacteria

### Take E.coli BL21 with plasmid pET28b for instance

#### Western blot

#### 【Materials】

#### ⒈ Apparatus:

Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane （Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.

#### ⒉ Reagents:

⑴ 10x buffer (660mL): Add 484.8g Tris , 2307.2g Glycine in ddH2O to final volume 660mL

  ⑵ 4x transfer buffer (5280mL):660mL 10x buffer, 3.3mL 20%SDS, 4620mL ddH2O

  ⑶ 1x transfer buffer (1 L): Add 200 ml Methanol, 250 ml 4x transfer buffer in ddH2O to final volume 1L

  ⑷ PBS buffer: Add 8g NaCl (137mM), 0.2g KCl (2.7mM), 1.44g Na2HPO4 (10mM), 0.24g KH2PO4 (2mM) to 1L ddH2O and adjust pH to 7.4 with HCl.

  ⑸ PBST buffer: 1L TBS buffer add 0.5ml Tween 20 (0.05%).

  ⑹ First antibody

  ⑺ Second antibody

  ⑻ 5% Blocking buffer (10ml): Add 0.5g Non-fat milk powde in PBST buffer to final volume 10ml, keep at 4°C to prevent bacterial contamination.

  ⑼ Developing reagent: Volume ratio: A reagent:B reagent=1:1.

  ⑽ Staining buffer: Add 1g amido black 18B (0.1%), 250ml isopropanol (25%) and 100 ml acetic acid (10%) to distilled water with final volume 1L.

  ⑾ Destaining buffer: Add 350ml isopropanol (35%) and 2 ml acetic acid(2%) to distilled water with final volume 1L.

#### 【Procedure】

#### ⒈ Separation of Protein

Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane （Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.

#### ⒉ Electrotransfer

⑴ Preparation of membrane

  Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10) according to the size of gel. Incubate in methanol for about 1 min on a rocker at room temp. Remove methanol and equilibrate membrane in 1x transfer buffer until ready to use.

  ⑵ Arrange gel-membrane sandwich

  In a shallow tray, open the transfer cassette. Put a well-soaked sponge pad on the black piece of the transfer cassette and a wetted 3MM paper on the sponge pad. Place the gel on the paper and arrange well so that all air bubbles are removed. Lay the PVDF membrane on the top of gel and remove any air bubbles. Place a wetted sheet of 3MM paper over the PVDF membrane and remove the bubble. Covered with the second well-soaked pad. Close the sandwich with the white piece of the cassette. Mount the sandwich in the transfer tank; put the black sides near the black side of the device. Fill the buffer tank with the transfer buffer.

  ⑶ Electrotransfer:

  Attach the electrodes. Set the power supply to 100V (constant voltage) for 1h at 4° C.

#### ⒊ Immunodetection

⑴ Membrane staining

  Disconnect transfer apparatus, remove transfer cassette, and peel 3MM paper from membrane. Remove the membrane to a small container. Add 10 ml PBST buffer and wash for short time. Cut out one stripe with 5mm width and put in another clean container. Stain this stripe in staining buffer for 1 min. Destain for 30 min in destaining buffer to check whether protein has been transferred from gel to membrane or not.

  ⑵ Membrane blocking and washing

  For other part of membrane, pour off PBST buffer. Add 5% blocking buffer，rock gently for at least 1 h. Pour off 5% blocking buffer and rinse briefly with PBST buffer three times, 5 minutes for per time.

  ⑶ First antibody

  Pour off PBST buffer. Add first antibody at appropriate dilution in 5 ml 0.5% blocking buffer. Rock gently for at least 1 h; pour off first antibody solution from membrane and wash twice for 10 minutes with PBST buffer.

  ⑷ Second antibody

  Pour off PBST buffer. Add second antibody at appropriate dilution in 5 ml 0.5% blocking buffer. Rock gently for 30min, pour off second antibody solution from membrane and wash twice for 10 minutes with TTBS buffer.

  ⑸ Detection

  Pour off PBST buffer from membrane and add developing reagent.Image with an imaging equipment.

#### Small plate reader for fluorescence

The procedure is similar with Standard curve. Protein can be replaced by the engineered bacteria.

#### Immunofluorescence detection

1. Take 200uL induced culture medium at 4℃, 12000rpm, centrifugation 1min. Retain the bacteria.

  2. Resuspend the recombinant yeast cells in PBS buffer, centrifuged at 12000rpm for 1 min at 4℃, and the supernatant was discarded. Retain the bacteria.

  3. The cells were resuspended in 200μL of 3mg/mL BSA in PBS buffer for 1h at room temperature. The cells were mixed up and down at 4℃, 12000rpm and centrifuged for 1min. Retain the bacteria.

  4. The cells were resuspended in 200uL of PBS buffer containing 3 mg / mL BSA containing 1μL of primary antibody.
Treat the cells overnight at 4℃, and the cells were allowed to mix up and down. The next day at 4 ℃, 12000rpm, centrifugation 1min. Retain the bacteria.

  5. Recombinant yeast cells were resuspended in PBS buffer, treated at room temperature for 10 min, centrifuged at 12000rpm for 1 min at 4 ℃. Retain the bacteria.

  6. Repeat step 5 twice.

  7. The cells were resuspended in 200uL of PBS buffer containing 1μL of secondary antibody and incubated for 1 h at room temperature. The cells were mixed up and down at 4℃ and 12000 rpm for 1 min. Retain the bacteria.

  8. The recombinant yeast cells were resuspended in PBS buffer for 5 min at room temperature. The cells were centrifuged at 12000rpm for 1 min at 4 ℃. The supernatant was discarded and retain the bacteria.

  9. Repeat step 8.

  10. Resuspended in 200uL of PBS buffer, each taking 10uL on clean static slide production.

#### Fluorescent examination in vivo

1. Incubate engineered bacteria overnight and induce them as shown above.

  2. Gather the cells and wish twice with PBS buffer. Resuspend them in 100 μL with 10^8 and 10^11 CFU, respectively.

  3. Get the experimental nude mice before the test.

  4. Use intraperitioneal injection to hocus the mice with 10% chloral hydrate.

  5. Use lavage needle to feed the mice with bacteria suspense.

  6. Keep photographing the mice in In-vivo Imaging System every 30 min for 6 hours.

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# Team:TJU China/Results

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## Results

#### [1. BV Detection](#collapseOne)

### 1.1 Standard curves

Our aim is to test the accurate amount of BV using smURFP we produced and purified. Then we get a series of standard curves of different concentration of BV and smURFP, as shown in the text-center below. Then we can use these curves to calculate how much BV is there in birds’ blood, or in other solutions.

![](https://static.igem.org/mediawiki/2017/a/a9/Tju_chn1.png)

![](https://static.igem.org/mediawiki/2017/8/89/TJu_chn2.png)

![](https://static.igem.org/mediawiki/2017/d/d7/Tju_chn3.png)

![](https://static.igem.org/mediawiki/2017/d/dd/Tju_chn4.png)

Form the results, we found that we can get good standard curves if we use more than 15μM (15μM included) protein for detection. And from these four curves, we got a final standard curve, as shown in the figure below.

![](https://static.igem.org/mediawiki/2017/7/7a/Tju_chn5.png)

Figure. Standard curves between concentration of BV and RFU. (concentration of smURFP should be more than 15μM)

### 1.2 BV production

![](https://static.igem.org/mediawiki/2017/f/f3/Tju_group1.png)

![](https://static.igem.org/mediawiki/2017/a/aa/Tju_group2%2Cpng.png)

The result after induction (with plasmid pET22a of HO-1 gene). The upper one is the control group, and the inferior one is the experimental group.

#### [2. Intestinal bacteria tracking](#collapseTwo)

### 2.1 molecular cloning

#### Escherichia coli BL21 & EHEC O157: H7:

a) Surface display - LPP

![](https://static.igem.org/mediawiki/2017/d/d5/Tju_pcr1.png)

PCR results for LPP. M is DNA marker. Lane 1 is the result
for LPP anchoring protein. Lane 2 is the result for smURFP
protein. Lane 3 is the result for overlap PCR.

b) Surface display – INPNC

![](https://static.igem.org/mediawiki/2017/3/35/Tju_pcr2.png)

PCR results for INPNC. M is DNA marker. Lane 1 is the result for INPNC
anchoring protein. Lane 2 is the result for smURFP protein. Lane 3 is the
result for overlap PCR.

c) Co-expression

![](https://static.igem.org/mediawiki/2017/5/57/Tju_pcr3.png)

PCR results for co-expression system. M is DNA marker. Lane 1 2 3 is the result for sumURFP protein. Lane 4 5 6 is the result for HO-1 protein. Lane 7 8 9 is the result for overlap PCR.

#### Citrobacter rodentium:

a) Surface display – INPNC

![](https://static.igem.org/mediawiki/2017/d/d4/Tju_pcr4.png)

PCR results for INPNC. M is DNA marker. Lane 1 2 3 is the result for smRUFP protein. Lane 4 5 6 is the result for INPNC anchoring protein. Lane 7 8 9 is the result for overlap PCR.

b) Surface display – Brka

![](https://static.igem.org/mediawiki/2017/b/b9/Tju_pcr5.png)

PCR results Brka. M is DNA marker. Lane 1 2 3 is the result for leading peptide and smURFP protein. Lane 4 5 6 is the result for Brka protein. Lane 7 8 is the result for overlap PCR.

c) Co-expression

![](https://static.igem.org/mediawiki/2017/7/77/Tju_pcr6.jpg)

Figure. Result of PCR for coexpression with promoter P2. M is marker. Lane 1 to lane3 are results for P2. Lane 4 to lane 7 are the results for smURFP. Lane 8 to lane 11 are the results for HO-1. Lane 12 to lane 14 are the results for P2-smURFP.

#### Bifidobacterium longum:

a) Surface display –GLBP

![](https://static.igem.org/mediawiki/2017/8/83/Tju_pcr7.png)

M is DNA marker. Lane is promoter HU, lane 2 is a meaningless sequence, lane 3 is smURFP, lane 4 is hu-meaningless sequence, lane 5 is Hu-meaningless sequence-smURFP, lane 6 is glbp

b) Surface display –AcmA

![](https://static.igem.org/mediawiki/2017/7/7d/Tju_pcr8.png)

M is DNA marker. Lane 1 is the PCR result for ACMA, Lane 2 is the plasmid of HU-meaningless sequence-smURFP, lane 3 is the digestion result of plasmid HU-ACMA-smURFP

c) Co-expression

![](https://static.igem.org/mediawiki/2017/0/03/Tju_pcr9.png)

M is DNA marker. Lane 1 is the PCR result of HU, Lane 2 is the PCR result of smURFP-histag, lane 3 is the PCR result HU-smURPF-histag, lane 4 is the PCR result of RBS-HO-1, lane 5 is the PCR result of HU-smURFP-histag-RBS-HO-1

### 2.2. Protein expression

#### Escherichia coli BL21 :

a) Surface display – INPNC

![](https://static.igem.org/mediawiki/2017/e/e6/Tju_pcr10.png)

The western blot result of INPNC in E.coli BL21. M is marker, Lane 1 is negative control. Lane 2 is INPNC-smURFP-His, which is about 40.4 kDa. Lane 3 is positive control.

b) Co-expression

| Tube number | 1 | | 2 | | 3 | |
| --- | --- | --- | --- | --- | --- | --- |
| Well number | 1' | 2' | 3' | 4' | 5' | 6' |
| RFU | 4315 | 4644 | 4783 | 5071 | 12 | 0 |

Result of Micro plate Reader in the black 96-well plate. Tube 1 and 2 are experimental group (with E.coli without plasmid), and tube 3 is the control group (with E.coli with plasmid pET28b of co-expression system).

### 2.3. Bacteria tracking in vitro

#### Escherichia coli BL21 - Co-expression

![](https://static.igem.org/mediawiki/2017/6/64/Tju_Confocal.jpeg)

Laser confocal microscopy was use to observe these bacteria directly. Excitation wavelength is 640nm.

### 2.4. Bacteria tracking in vivo

#### Escherichia coli BL21 - Co-expression

![](https://static.igem.org/mediawiki/2017/6/60/Tju_INVIVO.png)

The picture shows relative fluorescent intensity after doing intragastric administration (10^8 and 10^11 CFU) for 5.5h. The left is the control one (with E.coli without plasmid), the right is the experimental one (with E.coli with plasmid pET28b, 10^11 CFU). The result successfully showed that our system was executable and excellent. And smURFP has very compatible persistence and penetrability.

![](https://static.igem.org/mediawiki/2017/8/8c/Tju_chart.png)

Trend of RFU in vivo. M2 is the mice with 10^8 CFU bacteria, and M3 is the mice with 10^11 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 10^11 CFU bacteria.

#### [3. Protein structure](#collapseThree)

### 3.1. Protein purification

#### a) Nickel Column Purification

![](https://static.igem.org/mediawiki/2017/4/43/Tju_pcr11.png)

To test the purification efficiency and condition of Nickel Column Purification is via SDS-PAGE method. (M) The marker. (1) The 20 μl sample of E.coli crushing fluid before Nickel Column Purification with the target part of smURFP and his-sumo tag, around 26 kD. (2) The 20 μl sample of the solution with the target part is after Nickel Column Purification. (3)The 20 μl sample of the solution after is Nickel Column Purification is before the sumo protease digestion, with sumo protease around 13 kDa. (4) The 20 μl sample of the Nickel medium is after 12 h sumo protease digestion with the target protein smURFP, around 12 kDa. (5) The 20 μl sample of the eluent is with the protein smURFP. (6) The 20 μl sample of the Nickel medium after the elution to test whether the target protein is all in the solution.

#### b) Purification of ion exchange column & molecular sieve column.

![](https://static.igem.org/mediawiki/2017/5/58/Tju_chart1.png)

Result of purification of ion exchange column.

![](https://static.igem.org/mediawiki/2017/0/0f/Tju_chart2.png)

Result of purification of molecular sieve column.

![](https://static.igem.org/mediawiki/2017/4/49/Tju_pcr12.png)

To test the purification efficiency and condition of AKTA Ion Exchange Chromatography and Molecular Sieve Purification. (a) The 20 μl sample of the solution before Ion Exchange Chromatography Purification is as a compare. (b)~ (h) The 20 μl sample of the solution is after Ion Exchange Chromatography Purification. (i) The marker. (j) The 20 μl sample of the solution before Molecular Sieve Purification is as a compare. (l)~ (o) The 20 μl sample of the solution is after Molecular Sieve Purification.

### 3.2. Protein crystallization

![](https://static.igem.org/mediawiki/2017/9/90/Tju_Confocal2.png)

Result of proper condition for crystallization.

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