iGEM 2019 — Team JNFLS

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# Team:JNFLS/Notebook

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TEAM

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PROJECT

[Inspiration and Description](https://2019.igem.org/Team%3AJNFLS/Description)
[Design](https://2019.igem.org/Team%3AJNFLS/Design)
[Results](https://2019.igem.org/Team%3AJNFLS/Results)
[Experiments](https://2019.igem.org/Team%3AJNFLS/Experiments)
[Notebook](https://2019.igem.org/Team%3AJNFLS/Notebook)
[Demonstrate](https://2019.igem.org/Team%3AJNFLS/Demonstrate)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AJNFLS/Parts)
[Basic Parts](https://2019.igem.org/Team%3AJNFLS/Basic_Parts)
[Characterization](https://2019.igem.org/Team%3AJNFLS/Characterization)
[Improve](https://2019.igem.org/Team%3AJNFLS/Improve)

[MODEL](https://2019.igem.org/Team%3AJNFLS/Model)

HUMAN PRACTICES

[Human Practice](https://2019.igem.org/Team%3AJNFLS/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AJNFLS/Public_Engagement)
[Intended Application](https://2019.igem.org/Team%3AJNFLS/Hardware)

[SAFETY](https://2019.igem.org/Team%3AJNFLS/Safety)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=JNFLS)

# Notebook

## Week1 (4.8-4.14)

★Had the first meeting.

★Brainstorm and learning about relevant materials.

★Discuss our ideas and invited teachers to give some advice

## Week2 (4.22-4.28)

★Presentation to share what we learn about synthetic biology.

★Second Brainstorm.

★We began to use our spare time to study experimental techniques.

★project was decided.

## Week3 (5.12-5.11)

★We had a safety education before we started the formal experiment, including lab safety, researcher safety and environment safety. And we learned some emergency responses, such as how to use extinguisher and fire blanket, and how to use emergency shower and eye wash.

★Made a concrete plan for our project.

## Week4 (5.22-5.26)

★Extracting of plasmid.

★Constructing recombinant plasmid

★Learning cell culture techniques

## Week5 (5.27-6.2)

★The construction of recombinant plasmid.

★We have decided to build our parts and primers sequence was designed

★Transformation, monoclonal selected, we have sent plasmid to the company for sequencing verification

## Week6 (6.3-6.9)

★After connecting the cleavage products, we have sent them to the company for sequencing.

★We started building two parts using PCR technique and digestion technique:

★According to our design, We should know the sensitivity of the promoter. So we decided to construct two plasmids.

## Week7 (6.10-6.16)

★successfully constructed and cryopreserved.

★Continuing to build part.

★We went to Jinan Hing School to have teaching activities for publicity our project and Synthetic Biology.

★Preparing of experimental materials.

## Week8 (6.17-6.23)

★Construct part K3209000.

★Characterization K1334002

★designed a series of questionnaires.

★Data analysis.

## Week9 (6.24-6.30)

★It was verified by sequencing that we have building parts K3209000 successfully.

★Through a small range of issuing questionnaires, we have further modified our questionnaires.

★Continuing to construct other part, and preparing the materials for the second plasmid vectors.

## Week10 (7.1-7.7)

★Extracting plasmids and began to detect promoter sensitivity to formaldehyde.

★Exprssion theK3209000

★The construction the part K3209002.

★We started issuing questionnaires in a wide range.

## Week11 (7.8-7.14)

★Continuing to Construct the Part K3209003.

★SDS-PAGE analysis for exprssed protein K3209000.

★It was verified by sequencing that we have constructed the first vector successfully. And we began to constructed other part K3209006.

## Week12 (7.15-7.21)

★Continue to build part K3209004.

★Continue to expression protein, and purification.

★we have carried out preliminary experiments to be familiar with the use of instruments.

★Continue to construct the part.K3209006.

## Week13 (7.22-7.28)

★Expression and purification of expressed protein.

★It was verified by sequencing that we have building K3209002 successfully.

★we added a series of IPTG with a concentration gradient to some of the tubes with the same initial fluorescence intensity and measured the change in its fluorescence intensity.

## Week14 (7.29-8.4)

★Do HP work, publicity.

★The plasmid vector was finally constructed successfully after hard working, and we decided to transfect the plasmid into the cells.

★It was verified by sequencing that we have constructed the secong vector successfully.

## Week15 (8.5-8.11)

★Detected the sensitivity of promoter to fromaldehyde (repeat).

★We have made of poster for CCiC.

★After data processing, we got the graph which showed the influence of IPTG on the GFP expression.

## Week16 (8.12-8.18)

★Characterization the part: K3209006.

★Expression and purifation of protein (SDS-PAGE).

★Public propaganda of science in the Technology Museum.

★We transformed these two plasmids to competent cells, and according to their different resistances, we selected the strain of interest.

## Week17 (8.19-8.25)

★We take part in the CCiC in Shenzhen.

★We went to science museum to spread the idea of iGEM and hand out questionnaires.

## Week18 (8.26-9.1)

★SDS-PAGE for expressed the BFD-M7 and TalB-F187Y.

★Characterization part K1334002

## Week19 (9.2-9.8)

★We have launched a series of activities to promote the IGEM competition for Freshmen.

★A presentation for the students in our own school

## Week20 (9.9-9.15)

★Purification fo expressed protein.

★Characterization part K2890001

★HPLC analysis for products.

★Data analysis.

## Week21 (9.16-9.22)

★Repeat Purification fo expressed protein.

★Characterization part K2890001

★Repeat HPLC analysis for products.

★Data analysis.

## Week22 (9.23-9.29)

★Modify the condition of purification protein.

★Repeat HPLC analysis.

★Data analysis.

## Week23 (9.30-10.6)

★Characterization of K3209006.

★We have processed the data of questionnaire survey.

★After many measurements and data processing, we have realized the sensitivity of promoter to formaldehyde.

## Week24 (10.7-10.13)

★Start wiki work.

★Data analysis.

★Art work for wiki.

## Week25 (10.14-10.20)

★Data analysis.

★Continue wiki work.

★Prepared for Poster and presentation.


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# Team:JNFLS/Experiments

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TEAM

[Team Members](https://2019.igem.org/Team%3AJNFLS/Team)
[Attributions](https://2019.igem.org/Team%3AJNFLS/Attributions)
[Collaborations](https://2019.igem.org/Team%3AJNFLS/Collaborations)

PROJECT

[Inspiration and Description](https://2019.igem.org/Team%3AJNFLS/Description)
[Design](https://2019.igem.org/Team%3AJNFLS/Design)
[Results](https://2019.igem.org/Team%3AJNFLS/Results)
[Experiments](https://2019.igem.org/Team%3AJNFLS/Experiments)
[Notebook](https://2019.igem.org/Team%3AJNFLS/Notebook)
[Demonstrate](https://2019.igem.org/Team%3AJNFLS/Demonstrate)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AJNFLS/Parts)
[Basic Parts](https://2019.igem.org/Team%3AJNFLS/Basic_Parts)
[Characterization](https://2019.igem.org/Team%3AJNFLS/Characterization)
[Improve](https://2019.igem.org/Team%3AJNFLS/Improve)

[MODEL](https://2019.igem.org/Team%3AJNFLS/Model)

HUMAN PRACTICES

[Human Practice](https://2019.igem.org/Team%3AJNFLS/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AJNFLS/Public_Engagement)
[Intended Application](https://2019.igem.org/Team%3AJNFLS/Hardware)

[SAFETY](https://2019.igem.org/Team%3AJNFLS/Safety)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=JNFLS)

# Some experiments protocols

## Plasmid Extraction

1.Take out 1,500uL of cultivated bacteria liquid, which contained target plasmid, to t a 2ml micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 minute at room temperature, and then the supernatant was removed. Repeat this step three times to increase the concentration of the bacteria.

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

3.Add 250uL Solution II buffer, mixed evenly upside down 4-6 times; make the cell lysis solution fully, until the formation of bright. 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, gently and fully upside down 6-8 times, 12,000rpm centrifugation 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 Column. 12,000rpm centrifugation for 1 minute, the filtrate is removed.

7.Put the Column back into the collection tube, add 500uL HBC Buffer, 12,000rpm centrifugation for 1 minute, the filtrate is removed.

8.Put the Column back into the collection tube, add 700uL 75% ethanol, 12,000rpm centrifugation for 1 minute, the filtrate is removed. Repeat the wash step.

9.Put the Column back into the collection tube and 12,000rpm centrifugation for 1 minute.

10.Put the Column into a new 1.5mL micro-centrifuge tube and stand a while for 2-3 minutes. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 minute. 12,000rpm centrifugation for 1 minute. Discard the HiBind DNA Mini Column.

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

## Plasmid Extraction

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

1 µL DNA (concentration 1 µg/µL)

2 µL 10x buffer

1 µL each restriction enzyme (EcoR1 and Pst 1)

15 µL ddH2O

2.Incubate the reaction at digestion temperature (37°C) for 1 hour.

3.Stop the digestion 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 microcentrifuge 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 add 300µL of ADB).

9.Incubate 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 a Collection Tube.

11.Centrifuge for 30-60 seconds. Discard the flow-through.

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

13.Add ≥6µL DNA Elution Buffer or water directly to the column matrix. Place column into a 1.5 ml tube and 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 shown:

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 the temperature at 16°C for overnight.

## Preparing the Competent Cell

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

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

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

4.When the culture 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): LB medium 85% 8.5ml, PEG(wt/vol, Mw 8000) 10% 1.0g, DMSO(vol/vol) 5% 500μ l, MgCl2(pH 6.5) 50mM 500μ l.

## Transforming the cells

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

7.Heat shock at 42℃ for 2 minutes.

8.After heat shock, put on ice for 2 min; Add the cells into 0.8ml LB troth.

9.Shake and incubate at 37℃ for 60 minutes at 120 rpm.

10.Plate(＜200ul) on the appropriate agar plates with certain antibiotic.

11.Incubate the plates at 37℃ overnight.

## PCR experiment

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

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 machine.

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

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 your 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 shown:

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 trying not to get bubbles stuck underneath and allow another 30 min - 1 hour for complete polymerization.

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

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

9.Visualize the proteins using Coomassie Brilliant Blue.

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# Team:JNFLS/Results

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TEAM

[Team Members](https://2019.igem.org/Team%3AJNFLS/Team)
[Attributions](https://2019.igem.org/Team%3AJNFLS/Attributions)
[Collaborations](https://2019.igem.org/Team%3AJNFLS/Collaborations)

PROJECT

[Inspiration and Description](https://2019.igem.org/Team%3AJNFLS/Description)
[Design](https://2019.igem.org/Team%3AJNFLS/Design)
[Results](https://2019.igem.org/Team%3AJNFLS/Results)
[Experiments](https://2019.igem.org/Team%3AJNFLS/Experiments)
[Notebook](https://2019.igem.org/Team%3AJNFLS/Notebook)
[Demonstrate](https://2019.igem.org/Team%3AJNFLS/Demonstrate)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AJNFLS/Parts)
[Basic Parts](https://2019.igem.org/Team%3AJNFLS/Basic_Parts)
[Characterization](https://2019.igem.org/Team%3AJNFLS/Characterization)
[Improve](https://2019.igem.org/Team%3AJNFLS/Improve)

[MODEL](https://2019.igem.org/Team%3AJNFLS/Model)

HUMAN PRACTICES

[Human Practice](https://2019.igem.org/Team%3AJNFLS/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AJNFLS/Public_Engagement)
[Intended Application](https://2019.igem.org/Team%3AJNFLS/Hardware)

[SAFETY](https://2019.igem.org/Team%3AJNFLS/Safety)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=JNFLS)

# Results:

Our project intended to produce xylulose while removing formaldehyde indoors. In this process, two main genes are involved in our project, they are BBa\_K3209000 and BBa\_K3209002 parts which can express 2 important enzymes: BFD-M7 and TalB-F178Y.

BFD-M7 is Benzoylformate decarboxylase mutant from Pseudomonas putida. It contains 7 amino acids mutations, which sequence is different from BFD (BBa\_K2155001). The mutation information of BFD-M7 is as follows: W86R, N87T, L109G, L110E, H281V, Q282F and A460M. BFD-M7 can catalyze glycolaldehyde synthesis from formaldehye, then the glycolaldehyde reacts with formaldehyde to form dihydroxyacetone (DHA), also catalyzed by this enzyme.

TalB-F178Y is transaldolase mutant derived from E.coli, which is different from the part BBa\_K2155003, containing mutation F178Y. It catalyzes xylulose synthesis from glycolaldehyde and dihydroxyacetone (DHA). Along with the BFD-M7, TalB-F178Y catalyzes the xylulose production which is a precursor of some rare sugars.

![](https://static.igem.org/mediawiki/2019/5/5c/T--JNFLS--Results-1.png)

Figure 1. The pathway of xylulose synthesis from formaldehyde, including the synthesis of Glycolaldehyde and Dihydroxyacetone (DHA) from formaldehyde, catalyzed by BFD-M7, and the xylulose production, catalyzed by TalB-F187Y.

In order to achieve these two enzymes, we constructed the enzymes expression vectors: pET-28a-BFD-M7 and pET-28a-TalB-F187Y. And we identified these plasmids using electrophoresis, the results showed as Fig.2.

![](https://static.igem.org/mediawiki/2019/8/80/T--JNFLS--BasicParts-2.png)

Figure 2. Identification of BDD-M7(BBa\_K3209000) and TalB-F187Y (BBa\_K3209002) expression vectors construction. 1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker.

We also expressed these two enzymes in E.coli BL21, and purified them using Ni-NTA affinity chromatography. Results showed in Fig.3.

![](https://static.igem.org/mediawiki/2019/7/73/T--JNFLS--Results-3.jpeg)

Figure 3. Expression of BFD-M7and TalB-F187Y enzymes, and purified by Ni-NTA affinity chromatography. A. BFD-M7 expression and purification; B. TalB-F187Y expression and purification.

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. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7.

After success of expression and purification of BFD-M7and TalB-F187Y, we prepared the reaction system in which glycolaldehyde and dihydroxyacetone (DHA) from formaldehyde, followed by xylulose generation. HPLC was used to analyze the products catalyzed by BFD-M7and TalB-F187Y. Results showed in figure 4 and figure 5.

![](https://static.igem.org/mediawiki/2019/2/23/T--JNFLS--BasicParts-4.png)

Figure 4. HPLC analysis of the products catalyzed by BFD-M7.

A: Standard glycolaldehyde; B: Standard DHA; C: The products catalyzed by BFD-M7 in vitro. The results showed that our expression vector expressed active BFD-M7, which catalyzed the synthesis of glycolaldehyde and DHA.

![](https://static.igem.org/mediawiki/2019/4/40/T--JNFLS--BasicParts-7.png)

Figure 5. HPLC analysis of the xylulose product catalyzed by TalB-F187Y.

A: Standard Xylulose; B: The xylulose product catalyzed by TalB-F187Y in vitro. The results showed that our expression vector expressed active TalB-F187Y, which catalyzed the synthesis of xylulose from glycolaldehyde and DHA.

# References:

1.Bo Cui, Bingzhao Zhuo, Xiaoyun Lu, et al. Enzymatic synthesis of xylulose from formaldehyde. Chinese Journal of Biotechnology,2018, 34(7): 1128−1136.

2. Xiaoyun Lu, Yuwan Liu, Yiqun Yang, et al. Constructing a synthetic pathway for acetyl
coenzyme A from one-carbon through enzyme design. Nature communications, 2019 Mar 26;10(1):1378.