Team:TUST China/Experiments

Team:TUST China 2017

G. xylinus

G. xylinus Culture Media

G. xylinus
Broth Preparation(g/L)		G. xylinus
Agar Preparation (g/L)
Glucose25 25
yeast extract powder7.5 7.5
peptone10 10
Na2HPO4 20
Agar <20/td>
pH 6
Sterilization Autoclave 121℃ for 20min

G. xylinus Culturing

  1. Inoculum activation
    Harvest a loop of bacteria from Agar Slant using inoculation loop, inoculating it onto 50ml seed culture media (containing 4%（V/V） cellulolytic enzyme) in 250ml triangular flask. Incubate at 180 r/min，30℃, until its OD600 reach to 0.4~0.6, and save it as seed solution for later use.
  2. Preparation for seed solution
    Inoculate the seed solution into 200mL seed culture media (containing 4%（V/V） cellulolytic enzyme)in 500mL triangular flask. Incubate at 30℃，180r/min in shaker for 24 hours until the OD600 reach to 0.5.
  3. Flask-shaking Fermentation
    Following 4.5%(V/V) inoculum size, innoculate the seeds into the 200mL broth culture medium in 500mL triangular flask. Incubate at 30℃，180r/min in shaker for 120 hours.
  4. Stationary culture
    Following 4.5%(V/V) inoculum size, innoculate the seeds into the 200mL broth culture medium in 500mL triangular flask. Incubate at 30℃ in incubator.

Preparation for competence and Transformation of G. xylinus

Preparation of electro-competent G,xylinus cells(Imperial)

If the goal of transformation is to produce cellulose-producing transformed G.xylinus, use regular HS media for culturing. If cellulose production after transformation is not primary and speed is required, HS-cellulase medium can be used. Usage of HS-cellulase medium during growth results in the formation of a higher number of cellulose negative mutants, but results in much higher transformation efficiencies, and requires less time. Even with HS-cellulose medium, cellulose producing colonies can be identified on the plate after transformation, as cellulose-producing colonies differ in morphology from cellulose non-producing colonies (see section Gluconacetobacter for images of colony morphology).

Transformation of G.xylinus using electroporation

RNA extraction ofG. xylinus

Comparison between target strain and parent strain on fermentation performance.

under oscillating condition.

Inoculate a loop of overexpression strain, parent strain, and empty vector strain(control group) onto seed culture media respectively.Incubate them until OD600＝0.5. Following 4.5%（v/v）inoculum size, inoculate the strains onto fermentation media(containing 4%（v/v） cellulolytic enzyme) . Incubate at 30℃180rpm for 120h. Sampling and measure OD600, PH, residual sugar and gluconic acid for periodically.

under still condition.

Inoculate a loop of overexpression strain, parent strain, and empty vector strain(control group) onto seed culture media respectively.Incubate them until OD600＝0.5. Following 4.5%（v/v）inoculum size, inoculate the strains onto fermentation media(containing 4%（v/v） cellulolytic enzyme) Incubate at 30℃ ,let stew for 16 days. Sampling and measure OD600, PH, residual sugar and gluconic acid for periodically.

Methods of measuring these parameters.

  1. Measuring yield of cellulose

    Using 0.1M NaOH to remove the cells and residual media in cellulose, soaking it in water until pH reach to neutral. Dry to constant weight and weigh its mass.

  2. Measuring optical density of bacteria solution.

    Take 3mL fermenting liquid from each sampling points, use empty control group as zero setting. Utilize ultraviolet-visible spectrophotometer under 600nm to measure its OD.

  3. Measuring pH of fermentation solution.

    Take 3mL fermenting liquid from each sampling points, utilize pH meter to measure its pH and make sure it has been adequately shaken.

  4. Measuring residual sugar

    Take 3mL fermenting liquid from each sampling points, centrifuge 5000 r/min for 5 min. fetch the supernatant liquid. Dilute and measure the concentration of glucose in the liquid using SBA-40C biosensor.

  5. Measuring gluconic acid

    Preparation of chromatography: Aminex HPX-87H Strong acid cation exchange resin column, mobile phases 1Mm HClO4, flow velocity 0.5ml/min, column temperature 25℃,UV detector, wavelength 210nm, Injection amount 20μL , ESTD method.

Inoculate a loop of overexpression strain, parent strain, and empty vector strain(control group) onto seed culture media respectively.Incubate them until OD600＝0.5. Following 2.25%（v/v）inoculum size respectively, inoculate the strains onto the same fermentation media(containing 4%（v/v） cellulolytic enzyme) Incubate at 30℃ ,180 rpm in shaker for 120 h. Sampling periodically and dilution spread onto Amp agar plate and common agar plate. Cell counting until single colony appears.

Comparing the differences of cellulose mechanical property

  1. FT-IR

    Using Fourier Infrared Spectrum Technology to measure the structure and chemical bond of the samples in 4000-500 cm-1 wavelength.

  2. XRD

    Using X-ray diffractometer to measure crystal structure and crystallization properties etc. Stick the smooth surface of composite membrane on the sample board. Clip the sample board on the sample plate. Using TD-3500 X-ray diffractometer. This machine use Cu target. its scan interval is 0.006-96 °/min. Its operating current is 2-8 mA，1 mA/step. Its operation voltage is 40 K. Standard capacity is 5KW, the range of angular measurement is between -35-170 °. The accuracy is ≤0.0001 °. Test temperature is room temperature.

  3. Thermogravimetric analysis

    TGA analysis is to compare different material’s stability and tolerance in different temperature. The process is as follows :Take 10 mg samples, put it under the protection of nitrogen(250 mL/min), the temperature arise from 30 °C to 800 °C, the heating rate is 20 °C/min. Measure the degradation and loss of the materials during the overall warming. Before the TGA analysis, the sample should be preprocessed; dry to constant weight at constant 70°C.

E. coli / Co-culture

LB Broth Preparation(g/L)LB Agar Preparation(g/L)
peptone1010
yeast extract powder55
sodium chloride1010
Agar2%
pH7
SterilizationAutoclave 121℃ for 20min

Preparation for chemically-competence and Transformation of E.coli.

Preparation for chemically-competence and Transformation of E.coli.

  1. Inoculate a loop of fresh single-colony E.coli onto 25mL LB broth culture in 100mL triangular flask. Incubate at 180r/min, at 37℃ in shaker overnight.
  2. 2.Inoculate 1mL seed culture solution into 100mL LB broth culture media in 500mL triangular flask. Incubate 180 r/min, 37 ℃ in shaker until OD600 reach to 0.4-0.5
  3. 1.Transfer the incubated solution to two 50mL centrifugal tube respectively. Put these on ice for 30mins. Centrifuge 4000r/min for 10 min at 4℃ . Discard the supernatant.
  4. 4.Add 10 mL pre-cool calcium chloride and re-suspend the solution. Centrifuge 4000r/min for 10 min at 4℃ . Discard the supernatant.
  5. 5.Repeat step 4
  6. 6.Add 5mL pre-cool calcium chloride - glycerin into the centrifugal tube, re-suspend and transfer 100μL to 1.5mL sterilized EP tube.Thus the chemically-competent cell is ready.

General Heat-Shock Transformation

  1. Retrieve competent cells from -80℃.Add DNA to 50 ul cells on ice (no more than 5 ul, i.e. no more than 10% volume of cells)
  2. Incubate on ice 15-30 min(20min is recommended)
  3. Place the cells in iron bath to Heat shock 45℃ 90 s
  4. Place samples back on ice for 2 minutes
  5. Add 200 ul LB broth, or up to 10x volume of the cells
  6. Incubate at 37°C for 60 minutes, shaking
  7. Spin down cells（8000rpm 2min）, discard supernatant and resuspend in 100-200 ul LB to concentrate
  8. Plate out cells on LB agar(ampicillin 100mg/mL, x-gal 20mg/mL, IPTG 24mg/mL) , maximum 200 ul.
  9. Incubate upside down at 37°C for 24h， when blue-white spot appears , place in fridge 4℃ for half day.

General

PCR

PCR Reaction system

    1. Enzyme：2 x Taq PCR Mastermix
    Total volume25μL
    ddH2O 9.5μL
    Template 1 μL
    Forward primer 1 μL
    Reverse primer 1 μL
    2 x Taq PCR Mastermix 12.5 μL

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis

  1. After finishing PCR reaction , add 3 µL samples into gel pore .
  2. After the formation of gel, pull out the gel comb and take the gel out of the mould. Immerge the gel with 1×TAE buffer in the electrophoresis chamber.
  3. Using pipette to add marker and samples to different pore. (The content of pore depends on the gel comb, there are 3 kinds of volume, 10μL, 20μL, and 50μL) Do not stick the bottom and side of gel pore to prevent the leakage.
  4. Turn on the electrical source to start the electrophoresis, the voltage is set at 150-160V and the electrophoresis time is set at 8-12min..
  5. After the electrophoresis process end, the gel is observed under blue light or ultraviolet. Cut the DNA fragment from agarose gel under 365nm ultraviolet with a clean, sharp scalpel.
  6. Weigh the gel, measure its concentration and slice in recycling box.

Agarose Gel Electrophoresis Products Recycling

This protocol uses the commercially available SanPrep Column DNA Gel Extraction Kit.from Sangon Biotech(Shanghai) Co.,LTD. We have made some adaptation and for detailed user-guide and list of required reagent, see SanPrep Column Plasmid Mini-Preps Kit handbook.

  1. Preparation. add ethanol into Wash Solution;make sure Buffer B2 have no sedimentation; set the water bath to 50℃。
  2. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean tube. Add 3-6 time weight of the gel of Buffer B2 to the gel. Incubate at 50°C water bath for 5-10min until the agarose gel dissolves completely.
  3. Add 3-6 time weight of the gel of Buffer B2 to the gel. Incubate at 50°C water bath for 5-10min until the agarose gel dissolves completely.
  4. (Optional) If DNA fragment is <500 bp , it is recommended to add isopropanol which is 1/3 volume of Buffer B2 to the agarose gel sample after the gel completely dissolved
  5. Transfer the mixture gel to spin column. Centrifuge at 8000rpm for 30s.Discard the flow-through; place the Spin Column back into the collection tube again.
  6. Wash the Spin Column with 500 μl Wash solution and centrifuge at 9000 rpm for 30s. Discard the flow-through and place the Spin Column back into the collection tube.
  7. Repeat step 6
  8. Place the Spin Column back to the collection tube and centrifuge at 9000rpm for 1 min to remove residual wash buffer. Discard the flow-through.
  9. Transfer the Spin Column to a clean 1.5 ml microcentrifuge tube. Add 15-40μl volume of Elution Buffer to the center of the membrane, incubate at room temperature for 1 min, then centrifuge at 9000 rpm for 1 min.Store the DNA solution properly.

Note: The Elution Buffer can be replaced by 50μl water.

The linkage between target DNA fragments and vectors

Total volume 10μL
Target fragment 4μL
pMD18T 1μL
SolutionI5μL

Extraction of reconstructed plasmid and Verification of Restriction Endonuclease Cutting

Take 1-4mL overnight incubated bacteria solution, centrifuge at 12000r/min for 2 min. Use kit to extract.

Verification of reconstructed plasmids via single or double restriction digest

Total volumeDouble restriction digest(20μL)single restriction digest(20μL)singel restriction digest(20μL)
Water13μL14μL3μL
Plasmid3μL3μL2μL
Buffer2μL2μL
a1μL 1μL
b1μL
c1μL

Reaction time: 2h
Reaction temperature: 37℃
Restriction Endonuclease Cutting at 37℃ for overnight, undergo Agarose Gel Electrophoresis and wait for results.

Verfication of and construction of reconstructed plasmid(rubisco and others)

Total volumeDouble restriction enzyme digest(20μL)single restriction enzyme digest(20μL)
Water13μL14μL
Plasmid3μL3μL
Buffer2μL2μL
a1μL1μL
b1μL
c

Processing the product at 37℃ for 2 h, and undergo Agarose Gel Electrophoresis.

Expression of reconstructed plasmid

Expression reconstructed plasmid in E.coli

Transform the correctly-sequencing reconstructed plasmid into chemically-competence cell, select the transformants, extract its plasmid , verify through double enzyme digestion, and reserve the strain.
Select the mono-colony and inoculate onto 5mL LB Broth culture media in tube containing 100 μg/mL ampicillin.
Following 5% inoculum size, inoculate 200μL engineering bacteria into 20mL LB Broth culture medium(containing 100 μg/mL ampicillin) in 100mL triangular flask. Incubate at 37℃, 180 r/min in shaker until OD600 reaches to 0.4~0.6.
Add IPTG to 1 mM, incubate and induce 3h, 37℃，180 r/min. Collect the strain, add an adequate amount of milliQ and resuspend. Add 5×loading buffer, undergo water bath boiling and collect the supernatant to undergo protein electrophoresis for verification.

Synthesis of cDNA

DNA Purification

This protocol uses the commercially available SanPrep Column PCR Product Purification Kit from Sangon Biotech(Shanghai) Co.,LTD. We have made some adaptation and for detailed user-guide and list of required reagent, see SanPrep Column Plasmid Mini-Preps Kit handbook.

  1. Preparation. Add ethanol into Wash Solution;add isopropanol into Buffer B3; make sure Buffer B3 doesn’t appear sedimentation.
  2. Add Buffer B3(5 times the volume of PCR reaction solution) into PCR reaction solution.
  3. Centrifuge at 8000rpm for 30s.Discard the flow-through.
  4. Wash the Spin Column with 500 μl Wash solution and centrifuge at 9000 rpm for 30s. Discard the flow-through and place the Spin Column back into the collection tube.
  5. Repeat step 4 again
  6. Place the Spin Column back to the collection tube and centrifuge at 9000rpm for 1 min to remove residual wash buffer. Discard the flow-through.
  7. Transfer the Spin Column to a clean 1.5 ml microcentrifuge tube. Add 15-40μl volume of Elution Buffer to the center of the membrane, incubate at room temperature for 1 min, then centrifuge at 9000 rpm for 1 min.Store the DNA solution properly.

Note: The Elution Buffer can be replaced by 50μl water.