iGEM 2017 — Team TUST_China

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# Team:TUST China/Notebook

Team:TUST China 2017

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* PROJECT
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [Results](https://2017.igem.org/Team%3ATUST_China/Results)
    - [Optimization of
       Fermentation Conditions](https://2017.igem.org/Team%3ATUST_China/Ecoli)
    - [The Establishment of
       Co-Culture System](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
    - [Optimization of
       metabolic pathway](https://2017.igem.org/Team%3ATUST_China/Results/Gxylinus)
    - [BC Property Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* ACHIEVEMENT
  + [Achievement](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* NOTEBOOK
  + [Labbook](https://2017.igem.org/Team%3ATUST_China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ATUST_China/Experiments)
* PRACTICES
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Gold Integrated](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
  + [Silver](https://2017.igem.org/Team%3ATUST_China/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3ATUST_China/Engagement)
  + [iGEM Community](https://2017.igem.org/Team%3ATUST_China/HP/Community)
* TEAM
  + [Team](https://2017.igem.org/Team%3ATUST_China/Team)
  + [Attributions](https://2017.igem.org/Team%3ATUST_China/Attributions)

# Notebook

![](https://static.igem.org/mediawiki/2017/9/92/T--TUST_China--logo_note_ecoli.png)
![](https://static.igem.org/mediawiki/2017/9/96/T--TUST_China--logo_note_coculture.png)
![](https://static.igem.org/mediawiki/2017/a/a9/T--TUST_China--logo_note_more_BC.png)
![](https://static.igem.org/mediawiki/2017/6/6b/T--TUST_China--logo_note_application.png)

## *E.coli*

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the Results PDF file.](https://static.igem.org/mediawiki/2017/f/fd/T--TUST_China--notes_E_coli.pdf)

## Mixed Culture

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the Mix Culture PDF file.](https://static.igem.org/mediawiki/2017/3/3e/T--TUST_China--notes_mixed_culture.pdf)

## More BC

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the More BC PDF file.](https://static.igem.org/mediawiki/2017/3/32/T--TUST_China--notes_more_BC.pdf)

## Application

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the Results PDF file.](https://static.igem.org/mediawiki/2017/d/dc/T--TUST_China--notes_application.pdf)

* + [PROJECT](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [*G.xylinus*](https://2017.igem.org/Team%3ATUST_China/Results/Ecoli)
  + [Co-Culture](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
  + [BC Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* + [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* + [ACHIEVEMENT](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [PARTS](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* + [HUMAN PRACTICES](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Integrated and Gold](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
  + [Silver HP](https://2017.igem.org/Team%3ATUST_China/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3ATUST_China/Engagement)
  + [iGEM Community](https://2017.igem.org/Team%3ATUST_China/HP/Community)
* + [NOTOBOOK](https://2017.igem.org/Team%3ATUST_China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ATUST_China/Experiments)
* + [TEAM](https://2017.igem.org/Team%3ATUST_China/Team)
  + [Attributions](https://2017.igem.org/Team%3ATUST_China/Attributions)

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Tianjin University of Science & Technology

Address 1 : 1038 Dagu Nanlu, Hexi District, Tianjin, P.R.China (Postcode:300222)

Address 2 : NO.29 The Thirteenth Avenue Teda Tianjin, P.R.China (Postcode:300222)

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©2017 TUST IGEM. All Rights Reserved

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# Team:TUST China/Experiments

Team:TUST China 2017

[![](https://static.igem.org/mediawiki/2017/a/ad/T--TUST_China--Logo_2D.gif)](https://2017.igem.org/Team%3ATUST_China)

Toggle navigation

* PROJECT
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [Results](https://2017.igem.org/Team%3ATUST_China/Results)
    - [Optimization of
       Fermentation Conditions](https://2017.igem.org/Team%3ATUST_China/Ecoli)
    - [The Establishment of
       Co-Culture System](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
    - [Optimization of
       metabolic pathway](https://2017.igem.org/Team%3ATUST_China/Results/Gxylinus)
    - [BC Property Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* ACHIEVEMENT
  + [Achievement](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* NOTEBOOK
  + [Labbook](https://2017.igem.org/Team%3ATUST_China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ATUST_China/Experiments)
* PRACTICES
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Gold Integrated](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
  + [Silver](https://2017.igem.org/Team%3ATUST_China/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3ATUST_China/Engagement)
  + [iGEM Community](https://2017.igem.org/Team%3ATUST_China/HP/Community)
* TEAM
  + [Team](https://2017.igem.org/Team%3ATUST_China/Team)
  + [Attributions](https://2017.igem.org/Team%3ATUST_China/Attributions)

# Experience And Protocols

* [*G. xylinus*](#protocol-1)
* [*E. coli* / Co-culture](#protocol-2)
* [General](#protocol-3)
* Expand all
* Hide all

## *G. xylinus*

#### [*G. xylinus* Culture Media](#protocol-1-1)

|  | *G. xylinus*    Broth Preparation(g/L) | *G. xylinus*  Agar Preparation (g/L) |
| --- | --- | --- |
| Glucose | 25 | 25 |
| yeast extract powder | 7.5 | 7.5 |
| peptone | 10 | 10 |
| Na2HPO4 |  | 20 |
| Agar |  | <20/td> |
| pH | 6 |  |
| Sterilization | Autoclave 121℃ for 20min |  |

#### [*G. xylinus* Culturing](#protocol-1-2)

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*](#protocol-1-3)

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 of*G. xylinus*](#protocol-1-4)

#### [Comparison between target strain and parent strain on fermentation performance.](#protocol-1-5)

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.

#### [Competitiveness Experiment](#protocol-1-6)

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](#protocol-1-7)

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

#### [*G. xylinus* Culture Media](#protocol-2-1)

| LB Broth Preparation(g/L) | LB Agar Preparation(g/L) |  |
| --- | --- | --- |
| peptone | 10 | 10 |
| yeast extract powder | 5 | 5 |
| sodium chloride | 10 | 10 |
| Agar |  | 2% |
| pH | 7 |  |
| Sterilization | Autoclave 121℃ for 20min |  |

#### [Preparation for chemically-competence and Transformation of *E.coli.*](#protocol-2-2)

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](#protocol-3-1)

#### PCR Reaction system

1. Enzyme：2 x Taq PCR Mastermix

| Total volume | 25μL |
| --- | --- |
| 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](#protocol-3-2)

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](#protocol-3-3)

| Total volume | 10μL |
| --- | --- |
| Target fragment | 4μL |
| pMD18T | 1μL |
| SolutionI | 5μL |

#### [Extraction of reconstructed plasmid and Verification of Restriction Endonuclease Cutting](#protocol-3-4)

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 volume | Double restriction digest(20μL) | single restriction digest(20μL) | singel restriction digest(20μL) |
| --- | --- | --- | --- |
| Water | 13μL | 14μL | 3μL |
| Plasmid | 3μL | 3μL | 2μL |
| Buffer | 2μL | 2μL |  |
| a | 1μL | 1μL |  |
| b | 1μL |  |  |
| c |  |  | 1μL |

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)](#protocol-3-5)

| Total volume | Double restriction enzyme digest(20μL) | single restriction enzyme digest(20μL) |
| --- | --- | --- |
| Water | 13μL | 14μL |
| Plasmid | 3μL | 3μL |
| Buffer | 2μL | 2μL |
| a | 1μL | 1μL |
| b | 1μL |  |
| c |

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

#### [Expression of reconstructed plasmid](#protocol-3-6)

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](#protocol-3-7)

#### [DNA Purification](#protocol-3-8)

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.

* + [PROJECT](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [*G.xylinus*](https://2017.igem.org/Team%3ATUST_China/Results/Ecoli)
  + [Co-Culture](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
  + [BC Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* + [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* + [ACHIEVEMENT](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [PARTS](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* + [HUMAN PRACTICES](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Integrated and Gold](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
  + [Silver HP](https://2017.igem.org/Team%3ATUST_China/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3ATUST_China/Engagement)
  + [iGEM Community](https://2017.igem.org/Team%3ATUST_China/HP/Community)
* + [NOTOBOOK](https://2017.igem.org/Team%3ATUST_China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ATUST_China/Experiments)
* + [TEAM](https://2017.igem.org/Team%3ATUST_China/Team)
  + [Attributions](https://2017.igem.org/Team%3ATUST_China/Attributions)

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Tianjin University of Science & Technology

Address 1 : 1038 Dagu Nanlu, Hexi District, Tianjin, P.R.China (Postcode:300222)

Address 2 : NO.29 The Thirteenth Avenue Teda Tianjin, P.R.China (Postcode:300222)

[Home](https://2017.igem.org/Team%3ATUST_China)
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# Team:TUST China/Results

Team:TUST China 2017

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* PROJECT
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [Results](https://2017.igem.org/Team%3ATUST_China/Results)
    - [Optimization of
       Fermentation Conditions](https://2017.igem.org/Team%3ATUST_China/Ecoli)
    - [The Establishment of
       Co-Culture System](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
    - [Optimization of
       metabolic pathway](https://2017.igem.org/Team%3ATUST_China/Results/Gxylinus)
    - [BC Property Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* ACHIEVEMENT
  + [Achievement](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* NOTEBOOK
  + [Labbook](https://2017.igem.org/Team%3ATUST_China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ATUST_China/Experiments)
* PRACTICES
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Gold Integrated](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
  + [Silver](https://2017.igem.org/Team%3ATUST_China/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3ATUST_China/Engagement)
  + [iGEM Community](https://2017.igem.org/Team%3ATUST_China/HP/Community)
* TEAM
  + [Team](https://2017.igem.org/Team%3ATUST_China/Team)
  + [Attributions](https://2017.igem.org/Team%3ATUST_China/Attributions)

# Project Results

## Optimization of Fermentation Conditions

### Optimal pH

* During the fermentation process, problems like low efficiency of gene expression and low yield always occur. The formula of culture media and the condition of culturing in fermentation have direct or indirect relations with these issues. Particularly, pH can affect the dissociation of some certain components and intermediate metabolites ，which can influence the capacity utilizing these materials of bacteria. Meanwhile, the fluctuant environmental pH have certain impact on the pH level in the cells in the form of weak acid and alkali influencing the metabolic reaction of the cell. Therefore, an appropriate control of pH is crucial to the effective gene expression. That is the reason why we need to discover the optimal pH for the fermentation of *G.xylinus*.
  The scope of pH variation in the culture media during fermentation process is wide. In order to study the optimal pH for *G.xylinus* fermentation, we use acetic acid with 200 ionic strength to maintain the pH in the outside environment.

  The scope of pH variation in the culture media during fermentation process is huge. In order to study the optimal pH for *G.xylinus* fermentation, we use acetic acid with 200 ionic strength to maintain the pH value in the outside environment.

  | Initial pH | Buffer pH after ten days of fermentation | | | pH of the control group after ten days of fermentation | | |
  | --- | --- | --- | --- | --- | --- | --- |
  | 6.25 | 5.81 | 5.83 | 5.88 | 4.1 | 4.07 | 4.07 |
  | 5.75 | 5.58 | 5.64 | 5.45 | 4.06 | 4.1 | 4.06 |
  | 5.25 | 5.3 | 5.3 | 5.26 | 4.04 | 3.95 | 4.03 |
  | 4.75 | 4.84 | 4.83 | 4.76 | 3.99 | 3.88 | 3.98 |
  | 4.25 | 4.02 | 4 | 4.01 | 3.82 | 3.81 | 3.81 |

  ![](https://static.igem.org/mediawiki/2017/a/af/T--TUST_China--result_mix_G3.png)

  As you can see on the graph, the culture media with buffer in them has low pH variation. We have processed the membrane collected after 10 days of fermentation and the table below is the data we have collected.

  As the statistics show, when pH=5.75～5.25 ,the yield of BC the most optimal.Meanwhile, when pH=5.25 and 4.75, the yield of BC are higher than the control group by 11% and 7%.

  Therefore, we hope we can maintain the pH at 5 in the environment during *G.xylinus* fermentation.

  ![](https://static.igem.org/mediawiki/2017/2/2e/T--TUST_China--result_mix_G4.png)

### Transformation of *E.coli*

* Due to the production of acidic substances like glucuronic acid during *G.xylinus* fermentation, the accumulation of acidic compounds lead to the decrease in pH. In order to maintain the optimal pH for producing bacterial cellulose, a mechanism to produce alkali substances is required. We choose model strain *E.coli* considering its benefits such as having a clear genetic background, easy technical operation and short culture time. Therefore,we plan to modify *E.coli* to have the ability to respond to the variation of pH and enable it to produce alkali substances when the environment is acidic enough. Hence, the alkali substances produced can maintain the optimal pH for *G.xylinus* to produce BC.

  First, we need to find a switch with the function of sensing the pH level in the environment and respond accordingly. Luckily, we have identified a proton type of promoter P-asr located on the activator protein binding domain. When pH is around 5, it can activate RNA polymerase to combine with template DNA precisely resulting in the expression of downstream sequence. The pH for the activation of P-asr is very close to the optimal pH for producing BC. Therefore, we decide to use P-asr as our switch.

  After that, we have identified a gene GlsA with the function of producing alkali substances via the expression of glutaminase. This enzyme can catalyse the conversion of glutamine and water to glutamate and ammonia. The ammonia will combine with H+ to increase the pH in the environment.

  ![](https://static.igem.org/mediawiki/2017/1/16/T--TUST_China--result_mix_E1.png)

  We have verified its capability of producing alkali in DH5α

  ![](https://static.igem.org/mediawiki/2017/a/a0/T--TUST_China--result_mix_E2.png)

  Based on what we have acessed, we know that GlsA is activated when the pH is below 6. The graph above also demonstrates that the pH variation in the culture media of *E.coli* also fluctuate around 6.

  We add the gene of P-asr before the gene of glsa and construct a plasmid with pet28 as the backbone. We then transform it into *E.coli* DH5α and test the variation of pH during fermentation. The results is presented by the line graph below and we put it in comparison with the one with empty plasmid and the one with only GlsA. We can observe that the pH is maintaining at around 5.

  ![](https://static.igem.org/mediawiki/2017/7/7b/T--TUST_China--result_mix_E3.png)

  ![](https://static.igem.org/mediawiki/2017/4/41/T--TUST_China--result_mix_ph1.png)

  We put the transformed E.coli in co-culture with the transformed G.xylinus, detected the BC film they produced. The data are as follows.

### Control of Population Level

* We worry that the if the cell concentration of E.coli is too high, E.coli will compete with G.xylinus for nutrients leading to an negative impact on its growth. Therefore, we introduce Quorum sensing system and autophagy gene φx174E in E.coli.

  In order to control population levels , we engineered a synchronized lysis circuit (SLC) using cou- pled positive and negative feedback loops that have previously been used to generate robust oscillatory dynamics. The circuit(BBa\_K2267040) consists of a common promoter that drives expression of both its own activator (positive feedback) and a lysis gene (negative feedback).

  Specifically, the luxI promoter regulates production of autoinducer (AHL), which binds LuxR and enables it to transcriptionally activate the promoter. Negative feedback arises from cell death that is triggered by a bacteriophage lysis gene (φX174 E) which is also under control of the luxI promoter. AHL can diffuse to neighbouring cells and thus provides an intercellular synchronization mechanism.

  ![](https://static.igem.org/mediawiki/2017/f/f0/T--TUST_China--result_cp1.png)

  Quorum-sensing bacteria produce and release chemical signal molecules termed autoinducers whose external concentration increases as a function of increasing cell-population density.

  Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and alter gene expression, and therefore behavior, in response. Using these signal-response systems, bacteria synchronize particular behaviors on a population-wide scale and thus function as multicellular organisms.

  We modify the plux hoping to increase its sensitivity and we choose to conduct point mutation in the region of -10 according to the literature. Down below is the exact mutaion point and we add an indicator gfp after plux. We try to acquire a plux with higher sensitivity via modelling.

  ![](https://static.igem.org/mediawiki/2017/e/ef/T--TUST_China--result_cp4.png)

  ![](https://static.igem.org/mediawiki/2017/4/42/T--TUST_China--result_cp3.png)

  The bacterial population dynamics arising from the synchronized lysis circuit can be conceptualized as a slow build-up of the signalling molecule (AHL) to a threshold level, followed by a lysis event that rapidly prunes the population 。 After lysis, a small number of remaining bacteria begin to produce AHL anew, allowing the ‘integrate and fire’ process to be repeated in a cyclical fashion. We observe growth with the fluorescent protein superfolder GFP (sfGFP).

  ![](https://static.igem.org/mediawiki/2017/6/6d/T--TUST_China--result_cp2.png)

  ![](https://static.igem.org/mediawiki/2017/e/e6/T--TUST_China--part_part_results.png)

### Reference

1. A Glutamate-Dependent Acid Resistance Gene in Escherichia coli
2. Functional and Structural Characterization of Four Glutaminases from Escherichia coli and
   Bacillus subtilis
3. Glutaminase of Escherichia coli
4. Rational design of an ultrasensitive quorum-sensing switch
5. Genetic programs constructed from layered logic gates in single cells

## The Establishment of Co-Culture System

### Co-Culture :

* Based on what we have illustrated above, it is our priority to both improve the yields of cellulose generated from *G.xy* though exploring and optimizing the various condition of co-culture system during the fermentation process and make sure “engineered bacterial”——*E.coli* have grown stably and normally.

  Since the reproduction speed of *E.coli* have been much faster than the speed of *G.xy*, it is significant to calculate the ratio of inoculating both bacterial. Yields of cellulose will be compromised if we inoculate a number of *E.coli* while little volume of *E.coli* for which will fail to manipulate the pH.Therefore, it is necessary for us to to explore the best ratio of incubating *E.coli* and *G.xy*.

  During the process that we utilized culture medium for *G.xy* to fermented both *E.coli* and *G.xy*, the speed of forming the biofilm has been slowed down, therefore we went through the study of growth and metabolism of G.xy and noticed that the glycerol has played a crucial role in metabolic route of *G.xy*. According to the researches, adequate amount of glycerol is able to stimulate *G.xy* to secrete BC in the later phase. Based on what we have in mind, we decided to adjust the combination of carbon source of culture medium, which is turn the former pattern that glucose as the single carbon source to integrated carbon source pattern which use the glucose and glycerol to co-provide carbon source.

  In addition, we as well as did the trails to measure the impact of yields of BC from other impact factor as ethanol and citric acid etc..(Glu/Gly = Glucose/Glycerin)

  ![](https://static.igem.org/mediawiki/2017/8/81/T--TUST_China--result_mix1.png)

  We designed the table below and followed the steps to process our experiments, expiring different ratio of incubation and carbon source. Moreover, we dried the BC from day 7 culture medium and measured both the weight and thickness to obtain relative data.

  ![](https://static.igem.org/mediawiki/2017/0/07/T--TUST_China--result_mix2.png)

  The comparison of weight and thickness of BC showed that on the condition of equivalent carbon source and inoculated ratio of *G.xy* and *E.coli* is 4/1, the yields of BC will at least advance 11.5% compared to other groups. At the same time, we as well as discovered that the capacity of *G.xy* secrete BC has been significantly improved with the ratio of glucose and glycerol is 3/2~4/1.

  After setting up the inoculation level and the range of carbon ratio, we moved forward to optimize the carbon source through RSM. During this process, we collected a series of data which based on the yield as main target and the pH residue as co-factor, the data is present below:

  ![](https://static.igem.org/mediawiki/2017/a/a6/T--TUST_China--result_mix3.png)

  ![](https://static.igem.org/mediawiki/2017/6/6e/T--TUST_China--result_mix4.png)

  The finally optimize formula which has already reached the ideal ultimate pH and visibly improved the yields of BC is :

  | formula | Glu(g) | Gly(ml) | 50%EtOH | Predicting Final pH |
  | --- | --- | --- | --- | --- |
  | Target | 15.11 | 9.53 | 0.05 | 4.36114 |

  this optimized formula has similar carbon ratio to what we have measured in the culture medium of previous experiments, therefore we ultimately set up the carbon ratio as glucose/glycerol=3/2 of our co-culture medium.

  There is what we have gathered after utilized 3/2 medium in the co-culture system based on the former experiments:

  ![](https://static.igem.org/mediawiki/2017/b/b2/T--TUST_China--result_mix5.png)

  ![](https://static.igem.org/mediawiki/2017/4/4d/T--TUST_China--dty1.jpg)

  Compared the pH residue of single *G.xy* culture and co-culture during the fermentation, obviously approximately 15h after inoculation, pH of co-culture started to significantly drop while the yields of BC from co-culture clearly outnumber the yields of single *G.xy* culture.

  ### Reference :

  1. Factors affecting the yield and properties of bacterial cellulose
  2. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions
  3. Dynamic control and quantification of bacterial population dynamics in droplets

## Optimization of metabolic pathway

### Description :

* Cellulose can be synthesized by many organisms, ranging from prokaryote to animal. In prokaryotic organisms, bacterial cellulose is synthesized mainly by *Gluconacetobacter, Rhizobium, Agrobacterium, Escherichia, Enterobacter* and *salmonella*. In nature, only bacteria can synthesize cellulose pellicle with water-holding texture, among which the most studied bacteria are the genus Gluconacetobacter. Several common strains in the genus Gluconacetobacter can be distinguished by different representations. Bacteria that can synthesize bacterial cellulose are mainly *G. Europaeus, G. intermedius, G. Oboediens, G. Nataicola, G. Sacchari* and *G.xylinus*. Researchers have been studying the biosynthesis of bacterial cellulose in recent decades. This *G.xylinus* has long been recognized as a model strain. Many classical cellulose synthesis pathways and regulatory models were obtained in *G.xylinus*. Therefore,we choose *G.xylinus* as the research object in this experiment.

  The biosynthesis pathway of bacterial cellulose in *G.xylinus* is complicated including the synthesis of cellulose precursor, polymerization of glucan monomers and assembly of glucan chains.

  The biosynthesis of bacterial cellulose begins with the conversion of substrates to cellulose precursors, which are usually carbon sources, such as the conversion of glucose to uridine diphosphate glucose (udp-glucose). Uridine diphosphate glucose is the substrate of cellulose synthase, and it is catalyzed by cellulose synthase to form β-1 and 4 glucoside bonds between glucose monomers. There are specific sites on the outer membranes of the bacterial cells, which are coupled with the cellulose synthase complexes and is the spot where the polymerization is conducted [9]. When the polymerization reaction is over, the newly generated cellulose is secreted to the extracellular through these sites. The following figure is a partial schematic of the production of cellulose by *G. xylinus*

  ![](https://static.igem.org/mediawiki/2017/8/89/T--TUST_China--result_ecoli1.png)

  We hope to optimize the metabolic pathway of bacterial cellulose, redirecting more carbon sources into the production of bacterial cellulose; meanwhile expressing key genes in BC synthesis process. Through these two methods we hope to achieve high yield of BC . Therefore, we designed the following experiments:

---

### Design & Engineering :

* Phosphoglucomutase(PGM) is able to catalyze the conversion of glucose-1-phosphate and glucose-6-phosphate and plays an significant role in both glucose metabolism and BC metabolic network.

  Cyclic dimeric guanosine monophosphate (c-di-GMP) is a new type of second messenger molecule that is ubiquitous in bacteria. Bacteria utilize it to perceive cell surface signals and activate targets within cells, which involves amplification of the original signal and the subsequent expression of a series of specific genes in the cell. These genes affects the various physiological and biochemical processes in the cells. c-di-GMP control a wide range of process, and has a role in transcription, translation and post-translation levels. In addition, c-di-GMP participates in the regulation of multiple biological functions and affects biofilm formation. In a nutshell, it is a crucial elements to the formation of membrane-like bacterial cellulose and the improvement of BC production.

  This experiment attempts to improve BC production by over-expressing PGM and c-di-GMP, a small signal transfer molecule of bacteria.the goal is to implement both to realize the high yield of BC.

  The process of the experiment is divided into following steps, firstly, we transform the constructed plasmids into the E. coli DH5α. When expressing successfully, then we transform it into G.xylinus through electroporation and put it in comparison with the common G.xylinus. We try to explore the influence of each plasmids on the production of BC after fermentation.

  We use PCR to amplify gene PGM and c-di-GMP，and transform reconstructed plasmids J23119+PGM and J23119+c-di-GMP into G.xylinus. Then we put them in comparison with the control group PAsr+PGM and PAsr+c-di-GMP and measure its effect on the yield of BC respectively.

---

### Results

* **At the beginning** of the experiment, it was found that the yield of BC was higher than that of G.xylinus cultured individually when cultured with E.coli at pH=5 ，so we first construct the PPGM-PGM-331 plasmid and verify its effect on BC yield after overexpression.

  The results show that the overexpression of PGM has no significant effect on the increase of BC yield.

  1. We acquire genes of PAsr from K12 genome of *E.coli* via PCR
  2. We acquire genes of PGM and c-di-GMP from genome of *G.xylinus*.(With and without PGM)

     Construction of vector PPGM-PGM-pSEVA331. Transformation of plasmids into *E.coli*.

     Verification of homologous recombination via colony PCR

     Co-culture of *E.coli* and *G.xylinus*

     ![](https://static.igem.org/mediawiki/2017/5/59/T--TUST_China--result_ecoli2.png)
* **Second,** we found a strong promoter -J23119, linked it with the PGM, at the same time
  linked it with c-di-GMP which is a signal molecule could improve the yield of cellulose
  synthase.

  The results show that the yield of BC was not significantly increased, but the yield is increased compared to PPGM-PGM-331 plasmid, specially J23119-c-di-GMP.

  1. We acquire gene sequence J23119 and complete its linking with PGM and c-di-GMP
  2. We insert gene segments into plasmid pSEVA331 and transform it into chemically-competent *E.coli.*
  3. Verification of homologous recombination via colony PCR
  4. Co-culture of *E.coli* and *G.xylinus*

     ![](https://static.igem.org/mediawiki/2017/8/8f/T--TUST_China--result_ecoli4.png)

     ![](https://static.igem.org/mediawiki/2017/b/b1/T--TUST_China--result_ecoli42.png)

     ![](https://static.igem.org/mediawiki/2017/8/89/T--TUST_China--result_mix_dty2.png)
* Third, due to G.xylinus is co-cultured with E.coli in the environment of pH=5 as we
  designed, we also try to linked PGM with the acid-controlled promoter PAsr, and linked
  c-di-GMP with PAsr. We explore and compare the effects of overexpressing PGM and
  c-di-GMP on the yield of BC in the environment of co-culture and pH=5. At the same
  time,the effect of it and pH-responsive promoter PAsr on BC production will be
  compared.

  The results showed that the yield of BC was further improved than that of the second step, especially when PAsr was connected with c-di-GMP, and the yield of BC was significantly improved under mixed culture.

  1. We acquire gene sequence of PAsr＋PGM via PCR
  2. We acquire gene sequence of PAsr＋c-di-GMP via PCR
  3. We insert gene sequences into plasmid pSEVA331and transform them into chemically-competent cell into *E.coli*.
  4. Verification of homologous recombination via colony PCR
  5. Co-culture of *E.coli* and *G.xylinus*

     ![](https://static.igem.org/mediawiki/2017/6/6e/T--TUST_China--result_ecoli3.png)

     ![](https://static.igem.org/mediawiki/2017/7/7b/T--TUST_China--result_mix_E3.png)

     ![](https://static.igem.org/mediawiki/2017/3/33/T--TUST_China--result_mix_dty3.png)

---

### Reference

1. Peter Ross， Raphael Mayer， and Moshe Benziman. Cellulose Biosynthesis and Function in Bacteria [J] . Microbiological Reviews，1991，55(1):35-58
2. Peter Ross, Raphael Mayer, Haim Weinhouse, et al. The Cyclic Diguanylic Acid Regulatory System of Cellulose Synthesis in Acetobacter xylinum[J]. The J. of Biological Chemistry, 1990,265(31):18933-18943
3. Alan L. Chang, Jason R. Tuckerman, Gonzalo Gonzalez, et al. Phosphodiesterase A1, a Regulator of Cellulose Synthesis in Acetobacter xylinum, Is a Heme-Based Sensor[J].Biochemistry, 2001,40:3420-3426

## BC Property Testing

### Overview

* After the experiments above, we processed the bacterial cellulose film formed by the co-culture of the modified E. coli and G.xylinus, we would like to know whether this culture method of mixed bacteria will change the microstructure of BC? Therefore, we observed the morphological characteristics of the bacterial cellulose produced by the co-culture of the bacteria by scanning electron microscopy (SEM), infrared spectroscopy (FTIR) and atomic force. The results are as below: :

### 1. Scanning electron microscopy (SEM)

* We first used a scanning electron microscope to observe the formation of bacterial cellulose samples under different electric field strength. The results are shown in the figure

  ![](https://static.igem.org/mediawiki/2017/b/bf/T--TUST_China--project_mbc1.png)

  The microstructure, fiber detail and fiber width of the bacterial cellulose samples can be analyzed by scanning electron microscopy after freeze-drying. We found no significant difference between them.

### 2.Infrared Spectroscopy (FTIR)

* The use of Fourier transform infrared spectroscopy can help us understand the chemical structure of cellulose. Bacterial cellulose has an absorption peak for a particular wavelength of infrared, so that the composition of its chemical group can be analyzed.

  Respectively, we analyzed BC film under the two conditions after the natural drying by the infrared spectrum scan, the results shown in Figure. The absorption peak at about 1060 cm-1 reflects the vibration of the C-O-C group, which is the characteristic absorption peak of the cellulose. The absorption peak at 1428 cm-1 is caused by the bending vibration of the C-H bond, and the depth is related to the crystallinity of the cellulose. Although the infrared spectrum is a simple method, it can only give a relative value, it can show whether is a crystalline phase or non-crystalline phase at one time. It can be seen that the BC film produced under these two conditions are not much different in the composition of the structure.

  ![](https://static.igem.org/mediawiki/2017/c/c6/T--TUST_China--project_mbc2.png)

### 3. For the quality of wound dressings

* Bacterial cellulose is well suited for wound dressings due to its good biocompatibility, high mechanical strength in wet state, good liquid and gas permeability, and inhibition of skin infections. For secondary and tertiary burns, ulcers have been successfully used as temporary substitutes for artificial skin. For the application of bacterial cellulose in medical materials, our team visited the BC factory and learned more about what conditions are required for the products of BC films used as wound dressings.

  ![](https://static.igem.org/mediawiki/2017/4/47/T--TUST_China--project_mbc3.png)

  The above test content：| Test Items | Technical Requirements Clause | Technical Requirements | Test Result | Conclusion |
  | Water Content | 2.2.3 | ≦5 | 5 | qualified |
  | pH | 2.2.5 | 6.0±2.0 | 6.3 | qualified |
  | Heavy Metals | 2.2.6 | ≦10 | <10 | qualified |
  | Residue | 2.2.7 | <4.0 | 0.3 | qualified |

  At the same time we would like to thank xx company for providing the conditions of the test of our BC films . After a variety of tests of specifications, size, permeability, water content and others, data shows that the bacterial cellulose produced after the co-culture meet the technical requirements of wound dressings.

## Futurework

* During the experiments of overexpressing PGM, we discovered that it didn’t increase the yield of BC as expected. After researching relevant literature,we assume that due to the fact that G6P plays a significant role in the whole metabolic network, over expressing PGM may affect the direction of energy flux in the network. Therefore, the energy in the route of producing BC is going down leading to no increase in the yield of BC. On the other hand, perhaps the reaction PGM catalyzed is not a rate-limiting step or the overexpression of PGM hasn’t improve the efficiency of the enzymatic reaction. Other then that, it is very possible that there are still factors that we haven’t covered influencing the whole metabolic process and the yields of BC.

  Therefore.in the future, we might consider to modify or overexpress UGP which catalyze a rate-limiting reaction and explore its effect on the yields of BC

  Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties. So we want to make and characterise new cellulose binding domains, and fuse them to functional proteins. In our project, the bingding domains are the spytag and its catcher parts--spycatcher. We have designed a new spytag, including its RBS region, and make spytag link with GFP protein genes. We would verificate the catching power about this parts and do more experiments to give more functional properties into the bacterial cellulose.

  As we have increase the yield of bacteria cellulose fulfiling the protection of the membrance will wake the BC functional
  Since the BC membrance is accepted due to its mechanical properties，we would like to introduce another system to add new characteries to our product.
  According to the project of team Peking 2016 and resegrd papers we want to introduce the spytag-spychther system which is commonly used for binding. labeling immobilization and creating new kinds of application.
  We culture G.xylinus with the E.coli which can produce small peptid in the culture media evenly；thus， the spycather can be inlaid with BC membrance.
  The functional protein can be fused with spytag and be expressed bu the E.coli eddicieatly. We fuse the spytag with GFP.

* [Optimization of Fermentation Conditions](#section-1)
  + [Optimal pH](#section-1-1)
  + [Modification of *E.coli*](#section-1-2)
  + [Control of Population Level](#section-1-3)
  + [Reference](#section-1-4)
* [The Establishment of Co-Culture System](#section-2)
* [Optimization of Metabolic Pathway](#section-3)
  + [Description](#section-3-1)
  + [Design & Engineering](#section-3-2)
  + [Results](#section-3-3)
  + [Reference](#section-1-4)
* [BC Property Testing](#section-4)
  + [Overview](#section-4-1)
  + [SEM](#section-4-2)
  + [FTIR](#section-4-3)
  + [Quality](#section-4-4)
* [Futurework](#section-5)

* + [PROJECT](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Description](https://2017.igem.org/Team%3ATUST_China/Description)
  + [Design](https://2017.igem.org/Team%3ATUST_China/Design)
  + [*G.xylinus*](https://2017.igem.org/Team%3ATUST_China/Results/Ecoli)
  + [Co-Culture](https://2017.igem.org/Team%3ATUST_China/Results/Co_Culture)
  + [BC Testing](https://2017.igem.org/Team%3ATUST_China/Results/More_BC)
* + [MODELLING](https://2017.igem.org/Team%3ATUST_China/Model)
* + [ACHIEVEMENT](https://2017.igem.org/Team%3ATUST_China/Achievement)
  + [PARTS](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Safety](https://2017.igem.org/Team%3ATUST_China/Safety)
  + [Parts](https://2017.igem.org/Team%3ATUST_China/Parts)
  + [Collaborations](https://2017.igem.org/Team%3ATUST_China/Collaborations)
* + [HUMAN PRACTICES](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Summary](https://2017.igem.org/Team%3ATUST_China/Human_Practices)
  + [Integrated and Gold](https://2017.igem.org/Team%3ATUST_China/HP/Gold_Integrated)
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