1.Genomic

1.1 Extract genome

Add absolute ethyl alcohol to Buffer GD and Buffer PW before using them.

(1)Obtain 1~5ml bacterium sample in centrifuge tube, centrifuge at 10000rpm for 1 min and remove the supernatant.

(2)Add 200μl Buffer GA to the precipitate of bacteria, oscillate it to suspension.

(3)Add 20μl Proteinase K.

(4)Add 220μl Buffer GB, oscillate for 15 sec, place it at 70℃ for 10 min and centrifuge shortly.

(5)Add 220μl absolute ethyl alcohol, oscillate for 15 sec and centrifuge shortly.

(6)Transfer the obtained solution and precipitate from the step above to a Spin Column CB3, centrifuge at 12000rpm for 30 sec and discard the liquid.

(7)Add 500μl Buffer GD, centrifuge at 12000rpm for 30 sec and discard the liquid.

(8)Add 600μl Buffer PW, centrifuge at 12000rpm for 30 sec and discard the liquid.

(9)Repeat the eighth step.

(10)Put the Spin Column CB3 back into the collection tube, centrifuge at 12000rpm for 2 min and discard the liquid. Place it at room temperature for a few seconds to dry out the buffer.

(11)Transfer the Spin Column to a clean centrifuge tube, add 50~200μl Buffer TE to the middle of column membrane, place it at room temperature for 2~5 min, centrifuge at 12000rpm for 2 min and collect the solution to the centrifuge tube.

(12)Detect the concentration of DNA.

1.2 PCR

1. Make up a master mix of everything into eachPCR tube.

2. Run the " PCR" program, and adjust your extension time as described below:
Initial denaturation: 98°C for 5min
30cycles of:
95°C for 30 seconds
55°C for 30seconds
72°C for 30seconds
Final extension: 72°C for 10 min

2.Vector

2.1 Restriction Enzyme Digestion

To check if the two selected restriction enzymes can perform effective catalysis in the same solution
1. Mix DNA solution with the suitable amount of the master mix.

2. Pipette up and down in the EP tube.
        3. Incubate: 37°C for 30-40 min

2.2 Purification or Gel Extraction of DNA

2.2.1 Purification

Add 60ml ethanol (96-100%) to Buffer PW before use (see bottle label for volume). All centrifuge steps are in a conventional tabletop microcentrifuge at room temperature (15-25°C).

1. Column equilibration: add 500ul Buffer BL to the Spin Column CB2. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-throw, and then place Spin Column CB2 in the collection tube.

2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.

Note: For example, add 250μl of Buffer PB to 50μl PCR reaction (not including oil).

3. Transfer the mixture to the Spin Column CB2. Let it stand in refrigerator at 4℃  for 30 min. Centrifuge for 60s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-throw, and then place Spin Column CB2 back into the same collection tube.

Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.

4. To wash, add 600 μl Buffer PW to the Spin Column CB2 and place the Spin Column CB2 in refrigerator at 4℃ for 2 min, then centrifuge for 60s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB2 back in the same collection tube.

Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it suggests to let it stand for 2~5min after adding Buffer PW, and then centrifuge.

5. Wash the Spin Column CB2 with 600 μlBuffer PW and place the Spin Column CB2 in refrigerator at 4℃ for 2 min, then centrifuge for 60s at 12,000 rpm (~13,400 × g). Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer PW.

Note: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions.

6. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube and open CB2 lid and stay at 60℃ temperature for 20 min. To elute DNA, add 50 μl Buffer EB or deionized water (pH 7.0-8.5) to the center of membrane, let the column stand for 15 minat 60℃, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).

7. Alternatively, for increased DNA concentration, add the solution gained from step 6 to the center of membrane again, let the columns stand for 15 minat 60℃, and then centrifugefor 2 min at 12,000 rpm (~13,400 × g).

Note: The volume of Buffer EB must be more than 30 ul, or it may affect recovery efficiency. What’s more, the pH value of eluted buffer will have some influence in eluting, we suggest chose buffer EB or distilled water (pH 7.0-8.5) to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer AE and storing at –20°C is recommended, since DNA stored in water is subject to acid hydrolysis.

2.2.2 Gel Extraction of DNA

Column balance step:Place a TIANGEN DNA column CA2 in a provided 2 ml collection tube. Add 500μl Buffer BL to the TIANGEN DNA column, and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the TIANGEN DNA column back into the same collection tube.

Cut off the Single goal DNA band from the gel put it into the clean centrifuge tube and weigh.

Add same volume of Buffer PN more than the gel slice (0.1g gel account for 100μl). Incubate the mixture at 50°C for 10 min until the gel has completely melted. During the time we can mix it by shaking or overtaxing the tube.

Place a TIANGEN DNA column CA2 in a provided 2 ml collection tube. Add the mixture in last step to the TIANGEN DNA column, and place it at 4°C temperature for 30 min,then centrifuge at 12,000 rpm for 60 sec . Discard liquid and place the TIANGEN DNA column back into the same collection tube.

Add 600μl Buffer PW diluted with absolute ethanol into the TIANGEN DNA column and place it at 4°C temperature for 2 min and centrifuge at 12,000 rpm for 60 sec at room temperature to wash the column. Discard liquid and place the TIANGEN DNA column back into the same collection tube.

Note: Buffer PW Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, Buffer PW must be brought to room temperature before use.

Repeat the step 5.

Centrifuge the empty TIANGEN DNA column at 12,000 rpm for 2 min to dry the column, and place it at 60℃ temperature for 20 min. Do not skip this step, it is critical for the removal of ethanol from the TIANGEN DNA column.

Place the TIANGEN DNA column into a clean centrifuge tube. Add 50μl Buffer EB directly into the middle of the column membrane and place it at 60℃ temperature for 15 min. Centrifuge for 2 min at 12,000 rpm to elute DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

2.3 Ligation

1. Check the concentration of DNA fragments and vector which are going to be ligated.

2. Calculate the amount of part A/partB and vector added, based on the fragment length. Note that a ligation using a molar ratio of 1:3 vector to inserts.

3. Add DNA/buffer and ligase together in the EP tube.

4. Mix the reaction by pipetting up and down gently and microfuge briefly.

5. Incubate at 22°C for 1 hour or at 16°C for overnight.

2.4 Transformation (Competent cell: E.coli DH5α)

(1)Thaw DH5α cells on ice. Separate the cell into sterile precooling centrifuge tubes.

(2)Add 5µl of DNA to the cells. Mix the solution slightly and stand for 30 min on ice.

(3)Cells are incubated for 30 seconds at 42℃. Put cells back on ice quickly and stand for 2 min.

(4)Add 250µl LB broth(without antibiotics) to each centrifuge tubes. Incubate for 45 min at 37℃ on 150 rpm shaker.

(5)Spread 100µl onto the petric dish with LB agar(with antibiotics).

(6)Grow overnight at 37℃.

2.5 Colony PCR

1. Make up a master mix of everything into one microcentrifuge tube.

2.Run the "Simple PCR" program, and adjust your extension time as described below.
The "Simple PCR" program
Initial denaturation: 98°C for 5min
30cycles of:
95°C for 30 seconds
55°C for 30seconds
72°C for 35seconds
Final extension: 72°C for 10min

Plus.1 Luria Bertani Medium

Plus.2 DNA Agarose Gels

1. Prepare a 1% weight-to-volume agarose gel(take 100ml as example)

2. Dilute stock of 50×TAE to 1×with ddH2O.

3. Measure 100 ml of 1×TAE buffer.

4. Transfer 1×TAE buffer to Erlenmeyer flask.

5. Weigh out enough agarose to make 1% gel. (1% of 100mL is 1.0 g)

6. Transfer agarose to Erlenmeyer flask.

7. Melt agarose in microwave, stirring every 15-20 seconds until completely melted.

8. Allow gel to cool until Erlenmeyer flask can be handled comfortably. Then add 1:20 volume ratio (5μl) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.

9. Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.

Plus.3 Agarose Gel Electrophoresis

1. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).

2. Fill the rig with 1x TAE buffer.

3. Load 5µL of DNA maker into lane.

4. Mix 1µL of 6x loading buffer with 5µL DNA sample, load them into lane.

5. Run at 150V for 20 min.

6. Use Gel imaging system check gel.

7. Take picture for gel.

3.Preparation of competent

3.1 Competent  cell

1. Take bacterium fluid in 10 ml centrifuge tube, centrifuge it at low speed ( 4000rpm ),for 5min at 4℃.

2. Pour out on the clear liquid, by adding 5ml10 % glycerine solution, bacteria will weight new suspension. Then centrifuge it at the same speed and for the same time.
Repeat this step three times.

3. In the end, add 500μL 10 %glycerine solution to the bacteria sediment to make the residue resuspension.

4. Subpackage the bacteria solution to 5 EP tubes, so there is 100μLcompetent cell solution in each EP tube. Keep the competent cell solution in -80℃refrigerator.

3.2 Electrotransformation

1. Add 5μPCR product into 100μLcompetent cell solution, then put this mixture into 0.2cm electroporation cuvettes.

2. Then use BioRad Gene PulsenⅡ to electroporate our bacteria. Set the parameter as 25μF，2.5KV.

3. After electroporation add 1mL LB nutrient solution into our bacteria, the put this bacteria system in 37℃，150rpm incubator for 2 hours to removePIJ790 from our system.

4. Besmear the recoveryed bacteria liquidon the solid medium which has the corresponding resistance.

5. Culture the bacteria overnight at 37℃

4.Detection

4.1 Cell and protein OD

Take several bottles of liquid culture medium and add 5μL of bacteria in every one. Meanwhile, add 1μL of inducing agent in them except the control group. Placed the bottles in a shaking culture box.

After one hour, take out the liquid culture medium and shake them. Remove 2500μL bacteria under sterile conditions and add it in a clean cuvette, dilute it if necessary.

Adjust the absorbance of spectrophotometer of 600nm, then measure and record the ODs of the removed bacteria.

Remove 1500μL bacteria from the cuvette and add it in a clean EP tube, then centrifuge at 6300rpm for 10 min.

Extract supernatant 500μL and use it to rinse Syringe Filters, then take out the residual supernatant and add it in a new EP tube.

Remove 500μL supernatant and add it in a new clean cuvette, then add 2500μL shook Kaumas Coomassie brilliant blue G-250 and stand for 2 min.

Adjust the absorbance of spectrophotometer of 595nm, then measure and record the ODs of the csgA protein.

Repeat the step 2~7 until adequate data.

4.2 Polymerized curli fiber

1.add 5μL target glycerin bacteria to 100mL nutrient solution.Put  bacterium solution in 37℃，150rpmincubator , cultivate it for 7 hours.

2.Then add 1μL  1mol/LL arabinose to 100mL bacterium solution. Subpackage100mL bacterium solution into 10 test tubes averagely. Put subpackaged bacterium solution into 37℃Constant temperature and humidity incubator.

3.Take out each test tube as the schedule showed below.

10     20       20        30       30        30       30        60    /（min）

4.Filter the bacteria solution in each test tube.Use 700L PBS solution to make the residue resuspension. Add  0.8μLcongo red (10g/L) into 400μLdense medium, then water bath this system at 25℃ for 5 min.

5.In the end, centrifuge this system at 15000g centrifuge force for 5min, then measure the absorbance of the supernate.

June

We improved our initial idea and did more research about our project, including finding more information about properties of csgA and some particular application of this curli fibre. We made a rough schedule to arrange the experiment in the summer. And most of the protocols were established.

July

The first protocol we did was to extract the genome of our E.coli strains DH5a, then we designed several different primers in order to amplify the target gene sequences from the genome through PCR device. Those primers were designed to add traditional iGEM suffix and prefix on the initial sequences to introduce the restriction sites we need. The PCR product was confirmed by gel electrophoresis. CsgA and other necessary parts were confirmed and isolated and extracted. We attempted to ligate our sequence on the iGEM chloramphenicol backbone on July 10th. We need to add different promoters to those sequences and rearrange the orders of our parts. And there would be 4-5steps before we got our ideal plasmids. Each single step included ligation, transformation, confirming consequences and extracting plasmid of this step. The first step to construct these parts went well and we successfully added different promoters to the parts. However, we were trapped in the second step. We can’t efficiently isolated the parts using restrict enzyme. We finally found the reason on July 25th that we designed the wrong primers---we mixed the order of the four restriction sites when we design these primers. At the same time, we found that there was restrict site EcoRI inside the sequence of csgA and csgE, which need to be repaired. So we designed the new primers and started to reconstruct the parts we need on July 28th.

August

In august, our team started to reconstruct our parts. We designed to put our target gene into two different kinds of plasmids and transfer them into one BL21 cell. We put csgA, csgB into chloramphenicol backbone pSB1C3 with three kind of inducible promoters T7 , Pbad and Plam and two kind of constitutive promoters P1 and P2. We put csgE, csgF and csgG into another kind of backbone pSB3K3 with two constitutive promoters P2 and P3. We planned to construct 18 parts in total and completed several of them before august 30th.
Most E.coli strains have csgA in their own genome. Therefore, we began to knock out the csgA, csgB, csgC, csgE, csgF, csgG in the genome of backbone cell BL21 on august 25th

September

We constructed the rest of the biobricks before September 15th and then we began to transfer the E.coli BL21 we made with two different plasmids we had constructed and cultured the cells at the LB plates that contained both chloramphenicol and kanamycin to choose the colonies that were successfully transferred by two different plasmids. At the same time, we began to contact the instruments and devices we need. We designed a small device for csgA to properly assembling and we made our own Au electrode.

October

Tianjin University,Tianjin, China
Email:michaelss@tju.edu.cn