iGEM 2020 — Team JNFLS

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# Team:JNFLS/Notebook

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:JNFLS/Engineering

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Novel Coronavirus was first epidemic and first gain control in China. So we are lucky to have the opportunity to go into the lab and do research for our project. We have successfully cloned some genes involved in bacterial cellulose synthesis and transferred them into E. coli for obtaining more stable, higher-yielding bacterial cellulose. We have created some parts using these genes, and here are three of them.

## BBa\_K3605004

This part is Endo-β-1,4-glucanase(CMCax) with 1080bp, from Gluconacetobacter xylinus. It can hydrolyze the glucan chain and help straighten out the glucan, which is important for bacterial cellulose. When antibodies to recombinant CMCax are added to the culture medium, the formation of cellulose fibre is severely inhibited. This gene was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 1.

![](https://static.igem.org/mediawiki/2020/5/50/T--JNFLS--Engineering-1.png)

Fig.1. The result of CMCax gene cloning.

M: Marker; 1: PCR result of CMCax; 2: Digestion of pSB1C3 containing CMCax.

We used the software SnapGene Viewer to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows (figure 2):

![](https://static.igem.org/mediawiki/2020/4/4e/T--JNFLS--Engineering-2.png)

Fig.2. The sequence feature of CMCax gene.

## BBa\_K3605005

This part contains the CcpAx protein, also known as ORF-2, encoded by ccpax gene, functions as a mediator of protein-protein interactions and is important for localization of the bacterial cellulose synthase complex to the cell membrane in Gluconacetobacter xylinus. CcpAx gene, containing 936bp, was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 3.

![](https://static.igem.org/mediawiki/2020/f/fa/T--JNFLS--Engineering-3.png)

Fig.3. The result of CcpAx gene cloning.

M: Marker; 1: PCR result of CcpAx; 2: Digestion of pSB1C3 containing CcpAx.

We used the software SnapGene Viewer to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows (figure 4):

![](https://static.igem.org/mediawiki/2020/d/d3/T--JNFLS--Engineering-4.png)

Fig.4. The sequence feature of CcpAx gene.

CcpAx protein is crucial for the production of bacterial cellulose in the culture medium, because it helps the bacterial cellulose synthase to localize to the cell membrane. To make sure CcpAx protein is overexpressed in the transferred E. coli, we purified this protein using Ni-NTA affinity chromatography. The result indicated that CcpAx was successfully overexpressed, showed in the figure 5.

![](https://static.igem.org/mediawiki/2020/1/1c/T--JNFLS--Engineering-5.png)

Figure 5. Expression of CcpAx and purification by Ni-NTA affinity chromatography.

M: protein marker; 1: precipitation samples in the cell lysates; 2: supernatant samples in the cell lysates; 3: 50 mM imidazole eluent; 4: 100 mM imidazole eluent; 5: 200 mM imidazole eluent; 6: eluent after purification. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed CcpAx.

## BBa\_K3605006

This part is β-glucosidase encoded by bglxa gene with 2202bp in Gluconacetobacter xylinus. Both of the β-glucosidase and endoglucanase (CMCax, BBa\_K3605004) can hydrolyze tangled glucan chains when there is a failure chain arrangement and are both crucial for bacterial cellulose synthesis. This gene was also amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 6.

![](https://static.igem.org/mediawiki/2020/7/7b/T--JNFLS--Engineering-6.png)

Fig.6. The result of β-glucosidase gene cloning.

M: Marker; 1: PCR result of β-glucosidase; 2: Digestion of pSB1C3 containing β-glucosidase.

The software SnapGene Viewer was used to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows:

![](https://static.igem.org/mediawiki/2020/e/ee/T--JNFLS--Engineering-7.png)

Fig.7. The sequence feature of β-glucosidase gene cloning.

As we stated in the design of our project, there are 4 enzymes involved in the synthesis of bacterial cellulose starting with glucose. In addition, CcpAx protein helps the Bcs localize to the cell membrane; Endoglucanase (CMCax) and β-glucosidase can hydrolyze tangled glucan chains when there is a failure chain arrangement. Using overlapping PCR, all these genes are connected to form two polycistrons, which are inserted to the expression vector pETDuet-1. The identification result is showed in figure 8.

![](https://static.igem.org/mediawiki/2020/6/60/T--JNFLS--Engineering-8.png)

Fig.8. The gene cloning result of two polycistrons.

M: Marker; 1: Digestion of pETDuet-1 vector containing polycistron of GHK, PGM, UGPase, CMCax, CcpAx, and β-glucosidase; 2: Digestion of pETDuet-1 vector containing Bsc complex (BcsA, BcsB, BcsC, and BcsD).

After completion gene cloning work, the transferred E. coli were cultured in the medium for bacterial cellulose production. We used the optimized conditions acquired from the acetobacter xylinum culturing for producing bacterial cellulose. The E. coli were cultured for 7days at 30°C, pH6.8, 10% inoculum size, and glucose as the carbon source, then the cellulose in the medium was harvested. The result is showed in the figure 9.

![](https://static.igem.org/mediawiki/2020/b/b9/T--JNFLS--Engineering-9.png)

Fig.9. The production of bacterial cellulose in transferred E. coli.

The result showed that the bacterial cellulose was produced successfully in the transferred E. coli, indicating that the parts transferred to the E. coli worked well. The result also showed that the production yield is not as high as in the acetobacter xyilus (showed in the result of our project on the wiki), suggesting that the culture conditions still need optimization.

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# Team:JNFLS/Experiments

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# Key experiments protocols

## Plasmid DNA Extraction

1.Take out 1,500uL solution of cultivated bacteria that contained target plasmid to a 2ml micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 minute at room temperature, and then the supernatant was removed. In this tube, repeating these steps three times to increase the concentration of the bacteria.

2.Add 250uL Solution I buffer to suspend the precipitation. Suspension should be uniform, no small bacteria block left.

3.Add 250uL Solution II buffer, mix evenly upside down 4-6 times; make the cell lysis fully until the formation of bright vision. This step should not be more than 5 minutes, and avoid severe shock, so as to avoid contamination of genomic DNA.

4.Add 350uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000rpm for 10 minutes.

5.Put a HiBind DNA Mini Column into a 2mL Collection Tube.

6.The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000rpm for 1 minute, the filtrate is removed.

7.Put the Column back into the Collection Tube, add 500uL HBC Buffer, centrifuge at 12,000rpm for 1 minute, the filtrate is removed.

8.Put the Column back into the Collection Tube, add 700uL 75% ethanol, centrifuge at 12,000rpm for 1 minute, the filtrate is removed. Repeat the wash step once.

9.Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 1 minute.

10.Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2-3 minutes. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 minute. Centrifuge at12,000 rpm for 1 minute. Discard the HiBind DNA Mini Column.

11.Measure concentration of extracted plasmid DNA by nanodrop 1000, and store plasmid DNA at -20°C.

## Enzymatic Digestion of Plasmid DNA

1.Add components to a clean tube in the order as follow:

1 µL DNA (1 µg/µL)

2 µL 10x buffer

1 µL each restriction enzyme (EcoR1 and Pst 1)

15 µL ddH2O

2.Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 hour.

3.Stop the reaction by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.

4.The digested DNA is ready for use in research applications.

## Gel DNA Extraction

1.Buffer Preparation

2.Before starting:

3.Add 24ml 100% ethanol (26ml 95% ethanol) to the 6ml DNA Wash Buffer concentrate.

4.Add 96ml 100% ethanol (104ml 95% ethanol) to the 24ml DNA Wash Buffer concentrate.

5.DNA Wash Buffer included with D4001S and D4001T is supplied ready to use and does not require the addition of ethanol prior to use.

6.All centrifugation steps should be performed between 10,000 -16,000 xg.

7.Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it in to a 1.5 ml micro-centrifuge tube.

8.Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100µL (mg) of agarose gel slice adding 300µL of ADB).

9.Incubate it at 37-55℃for 5-10 minutes until the gel slice is completely dissolved.

10.Transfer the melted agarose solution to a Zymo-Spin™ Columnin Collection Tube.

11.Centrifuge for 30-60 seconds. Discard the filtrate.

12.Add 200µL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the filtrate. Repeat the wash step once.

13.Place the Column into a 1.5 ml tube, add ≥6µL DNA Elution Buffer or water directly to center of the column matrix, centrifuge for 30-60 seconds to elute DNA. Ultra-pure DNA is now ready for use.

## Ligation

1.Add components to a clean tube in the order as follow:

3 µL Promoter DNA

5 µL Plasma DNA

1 µL 10x buffer

1 µL T4 DNA Ligase

2.Mix them gently and fully, and incubate the reaction at 16°C for overnight.

## Preparing the Competent Cell

1.Streak the cell stock on a LB plate. Incubate the plate at 37°C overnight.

2.Pick a single well-isolated colony and inoculate it into 5mL of LB broth solution. Incubate it at 37°C overnight (more than 16 hours) with shaking at 220 rpm.

3.Transfer 250uL of the saturated overnight solution to a sterile 50mL polypropylene tube containing 25mL of LB medium. Incubate the cells at 37°C with shaking at 220 rpm until OD600 reaching 0.4. This usually takes 2.0-2.5 hour. Check the OD every 30 minutes after 1 hour to avoid overgrowth.

4.When the medium reaches an OD600 of 0.4, chill the tube on the ice for 30 min and then centrifuge at 8000rpm for 1minute at 4℃, discard the supernatant.

5.Resuspend the cells in 2.5mL of ice-cold TSS solution. Aliquote in 50uL per tube. Now the competent cells are ready to be transformed.

Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5ml, PEG(wt/vol, Mw 8000) 10% 1.0g, DMSO(vol/vol) 5% 500μ l, and MgCl2(pH 6.5) 50mM 500μ l.

## Transforming the cells

1.Add DNA (≤20uL) to ice cold 150uL competent cells. Incubate on ice for 30 minutes with occasional mixing.

2.Heat shock at 42°C for 2 minutes.

3.After heat shock, put it on ice for 2 min; Add the cells into 0.8ml LB medium.

4.Shake and incubate at 37°C for 60 minutes at 120 rpm.

5.Take out ＜200uL medium on the appropriate agar plates with certain antibiotic.

6.Incubate the plates at 37°C overnight.

## PCR experiment

1.Add components to a clean tube in the order as follow:

1 µL DNA Template

0.5uL Primer F

0.5uL Prmer R

10uL 2x Taq mixture

8uL ddH2O

2.Mix them gently and fully, and put the tube into a PCR instrument.

3.Set up the following PCR program: initial denaturing at 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 second, 55°C for 30 second, and 72°C for 60 second, plus a final extension at 72°C for 10 minutes.

4.Check the PCR product by agarose gel electrophoresis.

## SDS Gel Electrophoresis

1.make up 30 mL of running gel by adding components to a clean glass in the order as follow:

12.3 mL H2O

7.5 Ml 1.5 M Tris-HCl, pH 8.8

0.15 mL 20% (w/v) SDS

9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)

0.15 mL 10% (w/v) ammonium persulfate (APS)

0.02 mL TEMED

2.Mixing the ingredients shown above.

3.Pour the solution quickly into a gel casting form, and leave about 2 centimeters below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30 minutes for the gel to polymerize completely.

4.While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follow:

3.075 mL H2O

1.25 mL 0.5 M Tris-HCl, pH 6.8

0.025 mL 20% (w/v) SDS

0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)

0.025 mL 10% (w/v) ammonium persulfate (APS)

0.005 ml TEMED

5.Mixing the ingredients shown above.

6.Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to get bubbles stuck underneath and allow another 30 minutes - 1 hour for complete polymerization.

7.Preparing Sample: mix the protein 4:1 with the sample buffer. Heat the sample by Boiling for 5-10 minutes.

8.Running gel: clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.

9.Visualize the proteins using Coomassie Brilliant Blue.

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# Team:JNFLS/Results

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# 1.Gene cloning from Acetobacter xylinus

To realize the production of bacterial cellulose (BC) in E. coli, we cloned 6 genes related to the synthesis of BC from acetobacter xylinus. Some genes were amplified using PCR methods, and one of them was synthesized directly. In order to insert these genes to the bi-expressing vector pETDuet-1, which contains two separate promoters, some genes were connected together to form polycistron using the overlapping PCR method.

## 1.1 Glucose hexokinase (GHK)

This enzyme catalyzes Glucose converted to Glucose-6-phosphate (G-6-P), which is the glycolytic intermediate and the origin of substrate synthesis for bacterial cellulose production.

![](https://static.igem.org/mediawiki/2020/5/5c/T--JNFLS--Results-1.png)

Glucose hexokinase (GHK) was amplified using PCR method, and inserted into the vector pSB1C3. Figure 1 shows the identification result.

![](https://static.igem.org/mediawiki/2020/8/89/T--JNFLS--Results-2.png)

Fig.1. The result of Glucose hexokinase (GHK) gene cloning.

M: Marker; 1: PCR result of GHK; 2: Digestion of pSB1C3 containing GHK.

## 1.2 Phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase)

These two enzymes are original polycistron in the acetobacter xylinus, so we cloned them together. The phosphoglucomutase (PGM) can isomerize the glucose-6-phosphate (G-6-P) to glucose-1-phosphate (G-1-P), which is the second reaction for bacterial cellulose production from glucose. Another enzyme is UDP-glucose pyrophosphorylase (UGPase), it can catalyze the glucose-1-phosphate (G-1-P) to react with UTP, forming uridine-5’-phosphate-α-D-glucose (UDPG), which is the third reaction for bacterial cellulose production from glucose. The reactions catalyzed by them are as follows:

![](https://static.igem.org/mediawiki/2020/5/5c/T--JNFLS--Results-3.png)

These two enzymes were amplified using PCR method, and inserted into the vector pSB1C3. Figure 2 shows the identification result.

![](https://static.igem.org/mediawiki/2020/7/71/T--JNFLS--Results-4.png)

Fig.2. The result of PGM and UGPase gene cloning.

M: Marker; 1: PCR result of PGM and UGPase; 2: Digestion of pSB1C3 containing PGM and UGPase genes.

## 1.3 Bacterial cellulose synthase (Bcs) complex

Bacterial cellulose synthase (Bcs) complex contains 4 subunits BcsA, BcsB, BcsC and BcsD, which catalyzes the bacterial cellulose synthesis, using uridine-5’-phosphate-α-D-glucose (UDPG). This is the last reaction for bacterial cellulose production from glucose.

![](https://static.igem.org/mediawiki/2020/5/5e/T--JNFLS--Results-5.png)

Due to the long sequence (9102bp), this enzyme complex was synthesized directly and connected into pUC57 vector firstly, then it was inserted into bi-expressing vector pETDuet-1. The identification result is showed in Figure 3.

![](https://static.igem.org/mediawiki/2020/7/78/T--JNFLS--Results-6.png)

Fig.3. The gene cloning result of Bcs and other genes.

M: Marker; 1: Digestion of pETDuet-1 vector containing polycistron of GHK, PGM, UGPase, CMCax, CcpAx, and β-glucosidase; 2: Digestion of pETDuet-1 vector containing Bsc complex; 3: Digestion of pUC57 vector containing Bsc complex.

## 1.4 CcpAx protein (ORF-2)

The CcpAx protein, also known as ORF-2, functions as a mediator of protein-protein interactions and is important for localization of the Bcs complex to the cell membrane. This gene was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 4.

![](https://static.igem.org/mediawiki/2020/f/f3/T--JNFLS--Results-7.png)

Fig.4. The result of CcpAx gene cloning.

M: Marker; 1: PCR result of CcpAx; 2: Digestion of pSB1C3 containing CcpAx.

## 1.5 Endoglucanase (CMCax)

Endo-β-1,4-glucanase(CMCax) can hydrolyze the glucan chain and help straighten out the glucan, which is important for bacterial cellulose synthesis. When antibodies to recombinant CMCax are added to the culture medium, the formation of cellulose fibre is severely inhibited. This gene was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 5.

![](https://static.igem.org/mediawiki/2020/7/7b/T--JNFLS--Results-8.png)

Fig.5. The result of CMCax gene cloning.

M: Marker; 1: PCR result of CMCax; 2: Digestion of pSB1C3 containing CMCax.

## 1.6 β-glucosidase

β-glucosidase is encoded by bglxa gene in Gluconacetobacter xylinus. β-glucosidase and endoglucanase (CMCax) both can hydrolyze tangled glucan chains when there is a failure chain arrangement and are both crucial for bacterial cellulose synthesis. This gene was also amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 6.

![](https://static.igem.org/mediawiki/2020/3/3b/T--JNFLS--Results-9.png)

Fig.6. The result of β-glucosidase gene cloning.

M: Marker; 1: PCR result of β-glucosidase; 2: Digestion of pSB1C3 containing β-glucosidase.

## 1.7 Construction of polycistron

Polycistron is one of the characteristics in Prokaryote. It is common that some genes are regulated by the same promoter. So, some cloned genes were connected to form a polycistron using overlapping PCR in our project. Then this polycistron was inserted into the expression vector pETDuet-1. This process was identified in figure 7 and figure 8.

![](https://static.igem.org/mediawiki/2020/d/d5/T--JNFLS--Results-10.png)

Fig.7. The PCR results of GHK, (PGM and UGPase), CMCax, CcpAx, and β-glucosidase.

M: Marker; 1: GHK(969bp); 2: PGM and UGPase (2277bp); 3: CMCax(1080bp); 4: CcpAx(936bp); 5: β-glucosidase(2202bp).

![](https://static.igem.org/mediawiki/2020/2/2b/T--JNFLS--Results-11.png)

Fig.8. The overlapping PCR results of some genes.

M: Marker; 1: GHK + PGM + UGPase + CMCax + CcpAx + β-glucosidase (7464bp); 2: CMCax + CcpAx + β-glucosidase (4215bp); 3: CMCax + CcpAx (2013bp); 4: GHK + PGM + UGPase (3249bp).

At last, the two polycistrons containing all the gene required for bacterial cellulose production were cloned successfully into the expressing vector pETDuet-1, and transferred to the E. coli. Figure 9 shows the identification result.

![](https://static.igem.org/mediawiki/2020/0/0a/T--JNFLS--Results-12.png)

Fig.9. The gene cloning result of two polycistrons.

M: Marker; 1: Digestion of pETDuet-1 vector containing polycistron of GHK, PGM, UGPase, CMCax, CcpAx, and β-glucosidase; 2: Digestion of pETDuet-1 vector containing Bsc complex (BcsA, BcsB, BcsC, and BcsD).

# 2.The optimization of culture conditions of Acetobacter xylinus

When we were cloning these genes, we cultured the acetobacter xylinus to optimize some conditions for high BC production yield. This strain was donated friendly by iGEM team BNDS\_China, which collaborated with us all the time in this season.

We searched some references, and figure out that the temperature, pH value, fermentation time, inoculum size, and the carbon sources are the important conditions. So, we tried to culture the acetobacter xylinus for BC production with different conditions. And the results are showed in the following figures (Fig. 10-Fig. 14).

![](https://static.igem.org/mediawiki/2020/5/5c/T--JNFLS--Results-13.png)

Fig.10. The effect of temperature on BC production.

![](https://static.igem.org/mediawiki/2020/3/39/T--JNFLS--Results-14.png)

Fig.11. The effect of pH value on BC production.

![](https://static.igem.org/mediawiki/2020/8/8d/T--JNFLS--Results-15.png)

Fig.12. The effect of fermentation time on BC production.

![](https://static.igem.org/mediawiki/2020/b/be/T--JNFLS--Results-16.png)

Fig.13. The effect of inoculum size on BC production.

![](https://static.igem.org/mediawiki/2020/c/c0/T--JNFLS--Results-17.png)

Fig.14. The effect of carbon sources on BC production.

The optimization experiment results indicated that 30°C, pH6.8, fermentation 7 days, 10% inoculum size, and glucose serving as the carbon source are the best culture conditions for high BC production yield in acetobacter xylinus. And we know that for the BC production in E. coli, these conditions should be optimized again. But these optimized conditions can be referred for the following experiments.