iGEM 2020 — Team Nanjing-China

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# Team:Nanjing-China/Notebook

Notebook

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Notebook

NOTEBOOK

![](https://static.igem.org/mediawiki/2020/7/7d/T--Nanjing-China--notebook_timelline.jpg)

PROTOCOL

PolyP preparation

1. PolyP synthesis in bacteria

(1) Monoclonal streaking inoculation

The engineered bacteria containing the target plasmid (produced by the company according to the plasmid sequence and stored in a glycerol tube) were streaked on the Kana plate and cultured overnight at 37°C.

(2) LB culture

1) Prepared a 250ml Erlenmeyer flask, added 50ml LB, 50μl (1000x) Kana

2) Used a pipette tip to pick the monoclonal strain on the plate into an Erlenmeyer flask and shook it well. Cultured on shaker overnight at 37°C (12h).

3) Measured the absorbance of the bacterial solution at OD600 with LB zero adjustment, determined the amount of bacterial solution added to the synthetic wastewater culture medium, and ensured that the initial OD600 of the synthetic wastewater culture medium containing engineered bacteria was 0.2.

(3) Cultivation of synthetic wastewater

1) Centrifuged the bacteria-containing LB medium at 5000r for 1min, discarded the supernatant, resuspended it with a small amount of synthetic wastewater medium, pipette and mix well, centrifuged at 5000r for 1min, discarded the supernatant, and resuspended with a small amount of synthetic wastewater.

2) Prepared a 500ml Erlenmeyer flask, added 200ml synthetic wastewater culture medium, 200μl (1000X) Kana to each bottle, calculated the amount of synthetic wastewater culture solution after resuspension. Incubated on a shaker at 37°C for 12-14h.

(4) Determination of polyP content

1) Centrifuged at 12000r for 1min to separate the bacteria and the solution.

2) Took the supernatant and used the molybdate method to determine the phosphorus content in the solution. The reduced phosphorus was considered to be synthesized as polyP and stored in the bacteria.

2.Extraction of polyP

(1) Bacteria pretreatment

The recombinant engineered bacteria (containing polyP) cultured in synthetic wastewater for 14 hours were freeze-dried to obtain freeze-dried powder, and placed in a muffle furnace to heat at 600 degrees for 1 hour to obtain a carbonized black solid.

(2) Ground the black solid, added deionized water to dissolve it, the supernatant solution was the product solution containing polyP, placed it in a freeze dryer and freeze-dry to obtain the solid.

3. Purification of polyP (separation of polyP with different chain lengths)

(1) Used NaOH or KOH (2.5M) to adjust the pH of the polyP aqueous solution to 7, meanwhile, the total volume of the solution was V1

(2) In order to precipitate polyP with medium chain length, 0.156V1 volume of ethanol (96%v/v) was added to the solution and mixed well. The solution was stood for 1 hour to allow the polyP to fully aggregate together.

(3) Centrifuged the solution at 10000g for 5min after standing to separate the insoluble matter from the supernatant. The insoluble matter contained medium chain polyP (polymerization degree 30-40).

(4) Desiccation.opened the cap of the above centrifuged tube and placed it on a desiccator (a device filled with silica) for one week.

(5) Separated short-chain polyP, and collected the supernatant in step (3) into a new tube. At this time, the supernatant contained short-chain polyP (polymerization degree is about 11).

(6) Added 0.885V1 volume of absolute ethanol and mixed well. The solution was allowed to stand for 1 hour to allow the polyP to fully aggregate together.

(7) Centrifuged the solution at 10000g for 10min after standing, the insoluble matter contained short-chain polyP

(8) Desiccation, same as step (4).

**Attachments:**

**1. LB liquid medium formula**
Dissolved 0.5g of yeast powder, 1g of peptone, and 1g of NaCl in water and diluted to 100ml.
**2. Synthetic wastewater culture medium formula (100\* mother liquor, used sterile water to diluted and mix well**
(1) N source (high pressure steam sterilization)
Dissolved NaCl0.5g, MgSO4·7H2O2.36g, and NH4Cl1.8g in water, and diluted to 100ml.
(2) P source (high pressure steam sterilization)
Dissolved 1.48 g of dipotassium hydrogen phosphate trihydrate in water and diluted the volume to 100 ml.
(3) Y source (high pressure steam sterilization)
Dissolved 1g of tryptone and 0.1g of yeast extract powder in water and diluted to 100ml.
(4) Source C (filter sterilization)
Dissolved 3g glucose and 1.5g anhydrous sodium acetate in water and diluted to 100ml.
**2. Determination of phosphorus content in solution by molybdate method**
(1) Configuration of vitamin C and molybdate solution
1) Prepared 10g/100ml vitamin C solution and stored it in a brown bottle.
2) Dissolved 13g of ammonium molybdate tetrahydrate in 100ml of water and slowly added it to 300ml of 49% sulfuric acid. Dissolved 0.35g potassium antimony tartrate into 100ml water, slowly added and mixed well, stirring while adding. Stored in a brown bottle.

+36GFP preparation

1. Monoclonal streaking inoculation

The engineered bacteria containing the target plasmid (produced by the company according to the plasmid sequence and stored in a glycerol tube) were streaked on the Kana plate and cultured overnight at 37°C.

2. Monoclonal culture

(1) Prepared a 250ml conical flask, added 50ml LB, 50μl (1000x) Kana

(2) Used a pipette tip to pick the monoclonal strain on the plate into an Erlenmeyer flask and shook it well. Cultured on shaker overnight at 37°C (12h).

3. LB culture (purpose: protein expression)

(1) Prepared a 500ml Erlenmeyer flask, added 200ml LB, 200μl (1000X) Kana, 5ml monoclonal culture medium to each flask, and cultured at 37°C on a shaker.

(2) After transferring the last bottle, measured the OD600 of the first bottle, recorded the time and OD value

(3) Measured the OD every 2h. When the OD reached 0.1, bacterial would enter the logarithmic growth phase. After that, the OD measurement interval should be shortened appropriately. Estimated to reach the ideal OD value in 4h

(4) When the OD reached 0.6-0.8 (1.0 is fine), stopped shaking and added 1 mM IPTG to each bottle. (When the plasmid vector is PET29a (+), the amount of IPTG added was 1mM, and the configured IPTG concentration is 100mM, that was, 1000μL of 100mM IPTG was added to 100mL LB medium)

(5) Put it in a 37°C shaker and shake for 8-10 hours, and the medium turned green.

4. Collection and fragmentation of E. coli

(1) Collectd 1L of fermentation broth and centrifuged at 4℃, 6000rpm, 5min.

(2) Discarded the supernatant, resuspended the pellet, and transferred to a beaker.

Resuspended directly with high salt binding buffer: 1.5 M NaCl, 20 mM NaH2PO4-Na2HPO4 (pH 7.4),30 mM imidazole, 5 mM mercaptoethanol and protease inhibitor cocktail (Sigma-Aldrich).

(3) Bacteria breaking: ultrasonic breaking: placed the mixed bacteria in an ice box (without the liquid surface of the bacteria) and conducted ultrasonic breaking for 2s,
The interval was 3s, the power was 100%, and the alarm temperature was 20°C. Observe whether there were lumps in the solution until the bacteria become translucent.

5.Ni column purification

(1) Centrifuged the crushing liquid: 13000rpm, 4℃, 30min.

(2) Passed the supernatant through a 5mL HisTrap HP column (used a syringe or pump to push the pressure).

(3) Eluent elution (Eluent: 1.5 M NaCl, 20 mM NaH2PO4-Na2HPO4 (pH 7.4), 500 mM imidazole, 5 mM mercaptoethanol and protease inhibitor mixture).

(4) Diluted the collected eluate with cation exchange buffer 10 times (50 mM MES (pH 6.0), 1 mM DTT, 1 mM EDTA) to reduce the NaCl concentration to 150 mM.

(5) The diluted sample was passed through 5 mL HiTrapTM SP FF column (GE Healthcare), and the eluent was eluted (eluent: 50 mM MES (pH 6.0), 1 mM DTT, 1 mM EDTA, 1M NaCl, pH Adjust to 8).

(6) The eluate containing the target protein was concentrated and desalted by ultrafiltration (Using an ultrafiltration tube).

(7) Concentration and desalination using ultrafiltration centrifugation.

6.SDS analysis

The green fluorescent protein could be observed with naked eyes, which was used as a rough judgment. We used SDS-PAGE gel electrophoresis to perform accurate analysis based on the gel image.

**Attachments**

+36GFP combined with polyP

1. The search for phase separation

Mixed the solution according to the following concentration gradient, and searched for the phase separation boundary of the combination of polyP and +36GFP.

![](https://static.igem.org/mediawiki/2020/6/6c/T--Nanjing-China--notebook_picture_1.jpeg)

Determined the concentration of polyP and GFP for the binding experiment according to the phase separation boundary found, and selected the concentration in the phase separation boundary (main), boundary and outside of the boundary to set the gradient for the experiment.

2. The effect of the combination of polyP and +36GFP on the stability of GFP

(1) Mixed 5.56μM +36GFP and 10μM +36GFP with different concentrations of polyP in equal volumes, respectively incubated for 0h, 3h, 24h, 48h and then detected the fluorescence intensity.

(2) Mixed polyP (6400μM) and +36GFP (5.56μM) in equal volumes, in the phase boundary of liquid-liquid separation, respectively incubated at room temperature for 30min, then 25, 30, 40, 50, 60, 70, 80 Incubated for 10 min at 90, and 100℃ respectively. After cooling, detected the fluorescence intensity with a microplate reader.

(3) The final concentration of polyP (0,100,400,800,1600,3200μM) and the final concentration of +36GFP (2.78μM) protein were mixed in equal volume, respectively incubated at room temperature for 30min, and then incubated at 25,30,40,50,60,70,80,90 and 100℃for 10 minutes. After cooling, the fluorescence intensity was measured by a microplate reader.

(4) The final concentration of polyP (3200μM) and the final concentration of +36GFP (2.78μM) protein were mixed in equal volumes (50μl +50μl); the final concentration of +36GFP (2.78μM) protein was mixed in equal volumes with 20mM HEPES buffer (pH7), incubated at room temperature for 30 minutes, then respectively incubated with buffers of different pH (100 μl) for 30 minutes, and detected the fluorescence intensity with a microplate reader.

(5) +36GFP+polyP: polyP (0,200,800,1600,3200,6400) and +36GFP (5.56μm) mixed in equal volume (50μl +50μl), incubated at room temperature for 30min. After incubation, they were mixed with denaturing solution (100μl) and incubated for 30min. Microplate reader detected the fluorescence intensity.

**Attachments:**

**Denaturing solution**
1X denaturing solution; 4M urea, 0.5% SDS, 3M guanidine hydrochloride;
2X: 8M urea, 1% SDS, 6M guanidine hydrochloride.

About Spidroin & +36GFP-sp

The preparation method of spidroin and +36GFP-sp fusion protein was the same as +36GFP.

[NOTEBOOK #1](#cover_picture_div)

[PROTOCOL #2](#PROTOCOL)

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# Team:Nanjing-China/Engineering

Engineering

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[Design](https://2020.igem.org/Team%3ANanjing-China/Design)

[Engineering](https://2020.igem.org/Team%3ANanjing-China/Engineering)

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SOCIAL PRACTICES

[Human practices](https://2020.igem.org/Team%3ANanjing-China/Human_Practices)

[Education](https://2020.igem.org/Team%3ANanjing-China/Education)

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![picture display fail](https://static.igem.org/mediawiki/2020/6/68/T--Nanjing-China--cover.jpg "cover picture")
Engineering

We built three new parts:
**[[ Part:BBa\_K3341004 ]](http://parts.igem.org/Part%3ABBa_K3341004)** ,
**[[ Part:BBa\_K3341007 ]](http://parts.igem.org/Part%3ABBa_K3341007)** ,
**[[ Part:BBa\_K3341009 ]](http://parts.igem.org/Part%3ABBa_K3341009)** and proved they worked as expected.

We Completed A Synthetic Biology Cycle

We made an effort to follow the engineering design cycle:

![](https://static.igem.org/mediawiki/2020/c/ce/T--Nanjing-China--engneering_picture_2.png)

Design&Model: We simulated hybridization

At the suggestion of manager from Leqi textile, we mimicked strong electrostatic interactions in the binding between spidroin and polyP. Since the modeling parts indicated that amino acids like arginine, lysine and histidine contribute mostly to the electrostatic interactions, we tried similarly to mutate the primary sequence of spidroin to change its charges.

Build&Test: Spidroin Granules

Plasmid information

We changed the negatively charged amino acids in the non-helical and folded loop structures of spider silk proteins to lysine (the most positive amino acid) without affecting the main structure of the protein. It is positively charged to combine with the negatively charged polyphosphate through electrostatic force. In future experiments, the properties of spider silk proteins can also be improved through the combination of positively charged spider silk proteins and negatively charged inorganic molecules. Or through the fusion of spider silk protein and positively charged green fluorescent protein, the whole protein is positively charged, and at the same time, it combines the advantages of spider silk protein and green fluorescent protein, so that the position of this protein has green fluorescence. It can be clearly seen during separation or cell positioning, which is convenient for the observation of experimental results. It also has the good properties of spider silk protein. After combining with other organic and inorganic substances, it can be made into spider silk, which has good toughness and strength.

The negative charged amino acid in the non-helix and folded ring structure of spidroin was modified to lysine (the most positive electric amino acid) to make the spidroin positively charged without affecting the main structure of the protein.
SP1 mainly modifies N-terminal and C-terminal.

Plasmid function verification

In subsequent experiments, we combined the spider silk protein green fluorescent protein with polyphosphate, which has better thermal stability, PH stability and better mechanical resistance. The spider with positive electricity Silk protein can be easily and tightly combined with negatively charged molecules, which is convenient for studying the physical and chemical properties of different organic-inorganic hybrid materials. In the experiment of purifying spider silk protein, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify spider silk protein (to achieve the purification of positively charged protein). The SDS-PAGE detection results of the protein purified by the above two columns are shown in the figure:

![](https://static.igem.org/mediawiki/2020/9/92/T--Nanjing-China--engineering_picture_1.jpeg)

Figure 1 spidroin SDS-PAGE test result

Remarks: BL21 refers to the transfer into the empty PET29a vector without the target gene, and the specific whole protein is obtained by inducing expression.

![](https://static.igem.org/mediawiki/2020/3/3e/T--Nanjing-China--experiment_picture_3.jpeg)

Figure 2 purified +36GFP、spidroin SDS-PAGE test result

As shown in Figure 1, in the whole protein, Sp and Sp1 have bands around 40KDa, indicating that Sp, Sp1 and Sp2 can all be expressed in E. coli. Among them, Sp2 has a more obvious band around 40KDa, indicating The expression level of Sp2 in E. coli is relatively large. In the precipitate, Sp2 appeared as a band around 40KDa, but there was no corresponding band in the supernatant, indicating that Sp2 was mainly present in the precipitate. The bands of Sp and Sp1 are not obvious in the whole protein and supernatant, indicating that the expression level is low.

In order to further verify the expression of Sp, Sp1 and Sp2, we performed SDS-PAGE again. As shown in Figure 2(A), the whole protein of Sp2 appears in a corresponding band near 40KDa, indicating that Sp2 can be expressed in the bacteria. In the precipitation, Sp2 showed a band near 40KDa, but there was no band near 40KDa in the supernatant, indicating that Sp2 protein mainly exists in the precipitate. As shown in Figure 2 (A), the supernatants of Sp and Sp1 have bands around 40KDa, indicating that Sp and Sp1 can also be expressed in the bacteria, but the expression level is low and the column hanging effect is poor.

Learn: Learnt from interviews with Prof. Tian&Prof. Cao

We interviewed Mr. Cao and Mr. Tian from College of engineering and applied sciences. Prof. Tian gave us suggestions on protein purification and stressed the importance of buffer for successful protein elution. Besides, professor Cao pointed out that reconstructing spidroin may affect its folding. Therefore, he proposed us a new idea and inspired us to combine spidroin with + 36GFP to form a new fusion protein, and then hybrid the fusion protein with polyP.

Redesign&Rebuilt: We built a new part and proved it worked as expected

Inspired by these professors, we tried to fuse GFP and spidroin.

We attached +36GFP-SP fusion protein gene sequence to PET28a vector and obtained the recombinant plasmid PET28a-+36GFP-SP, which was transformed to the competent cell BL21 (DE3) and induced by 1M IPTG at 25℃ overnight for later protein purification. The protein purification result shows that +36GFP-SP fusion protein produced by recombinant plasmid PET28a-+36GFP-SP can interact with Ni column and led to the successful purification. The SDS-PAGE detection results of the protein purified by the two kinds of columns are shown in the figure below:

![](https://static.igem.org/mediawiki/2020/2/2e/T--Nanjing-China--engneering_picture_3.png)

Figure 3 Result of +36GFP-SP SDS-PAGE

In reference to figure 3, bands of 70kDa appeared in both total protein track and supernatant track, while no band of 70kDa appeared in the precipitation track. This indicates that +36GFP-SP fusion protein gene was successfully expressed by BL21 and most +36GFP-SP fusion protein existed in supernatant after cell disruption, which made it of value for the following purification step. Moreover, combined with the electrophoresis results of +36GFP-SP after purification in Figure 1, a weak band near 70kDa appeared, which further indicated the success of +36GFP-SP purification.

More experiments are still continuing.

More information about our experiments

Related plasmids synthesized by polyP

Plasmid information

Natural function of part: The polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracellular inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain.

Wild-type Citrobacter freundii ATCC 8090 was purchased from China Center of Industrial Culture Collection (CICC, China). The ppk1 gene was acquired by PCR. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.

Although PPKs from E. coli and C. freundii shares 96% amino acids’ identity, the C. freundii PPK has a glutamate and a lysine residue in positions 327 and 328, where in E. coli they are substituted by much less strongly charged alanine and glutamine residues, respectively. Although these natural occurred mutation sites found in C. freundii PPK are distant from the enzymes’ active site, they lie in the interfaces among monomers of the PPK tetramer. Benefit from this difference, a dramatic increase of intracellular polyP accumulation can be achieved with C. freundii.

Plasmid function verification

The polyP plasmid is highly expressed in Citrobacter fujii. It is inoculated into wastewater culture medium after 12 hours of expanded culture in LB medium. The bacteria can use the inorganic phosphorus in synthetic wastewater to synthesize polyphosphate, the largest The output is about 20mg/L. Our plasmid is resistant to kanamycin, so it grows predominantly in LB medium supplemented with kanamycin. At the same time, it is the only predominant species in wastewater medium due to the scarcity of nutrients, but in wastewater medium The number of reproduction is not much and more energy is used to synthesize polyphosphate, and the yield is the highest 14 hours after inoculation, and the prolonged time may lead to a decrease in yield and the decomposition of polyphosphate. Among them, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest system output and the highest conversion efficiency. Almost all inorganic phosphates can be converted into polyphosphates under the condition of no other bacteria pollution and normal bacterial viability. Finally, we obtained 839.28mg of polyP in the bacteria through bacterial synthesis; after extraction, we obtained 489.89mg of pure polyP; through the ethanol separation method, we finally obtained 214.67mg of polyP 45 with suitable chain length that can be used in subsequent binding experiments.

+36GFP plasmid

Plasmid information

The nucleotide sequence of GFP (+ 36) was biosynthesis of Qingke, using PUC57 with ampicillin resistance. The N-terminal restriction site was KPN I, the C-terminal site was hind III, and the host cell was E.coli Top 10. GFP green fluorescent protein (GFP) is improved by adding 36 positive charges, making it easier to combine with negatively charged proteins through electrostatic force. Because GFP has intrinsic green fluorescence, it is easier to be located in cells and tissues, not only can determine the distribution of negatively charged proteins, but also, to a certain extent, can indicate the electrical properties and structural changes of binding proteins. Positively charged green fluorescent protein (GFP) provides negatively charged molecules with a way to change their electrical properties, bind to negatively charged molecules and label negative proteins.

Plasmid function verification

After comparison and verification, the synthesized nucleotide sequence is translated into protein, which is consistent with the expected sequence. We ligated the +36GFP target gene sequence to the PET29a vector to obtain the recombinant plasmid PET29a-+36GFP. The recombinant plasmid was transformed into the competent cell BL21 (DE3), and the final concentration of 1mM IPTG was induced at 16°C overnight for subsequent protein purification. In the experiment of purifying +36GFP, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify +36GFP(to achieve the purification of positively charged protein). The SDS-PAGE detection results of the protein purified by the above two columns are shown in the figure:

![](https://static.igem.org/mediawiki/2020/6/6a/T--Nanjing-China--experiment_picture_2.jpeg)

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# Team:Nanjing-China/Experiments

Experiments

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[HOME](https://2020.igem.org/Team%3ANanjing-China)

TEAM

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PROJECT

[Description](https://2020.igem.org/Team%3ANanjing-China/Description)

[Design](https://2020.igem.org/Team%3ANanjing-China/Design)

[Engineering](https://2020.igem.org/Team%3ANanjing-China/Engineering)

[Experiments](https://2020.igem.org/Team%3ANanjing-China/Experiments)

[Model](https://2020.igem.org/Team%3ANanjing-China/Model)

[Notebook](https://2020.igem.org/Team%3ANanjing-China/Notebook)

[Implementation](https://2020.igem.org/Team%3ANanjing-China/Implementation)

[Contribution](https://2020.igem.org/Team%3ANanjing-China/Contribution)

[PARTS](https://2020.igem.org/Team%3ANanjing-China/Parts)

[SAFETY](https://2020.igem.org/Team%3ANanjing-China/Safety)

SOCIAL PRACTICES

[Human practices](https://2020.igem.org/Team%3ANanjing-China/Human_Practices)

[Education](https://2020.igem.org/Team%3ANanjing-China/Education)

[Collaborations](https://2020.igem.org/Team%3ANanjing-China/Collaborations)

[Partnership](https://2020.igem.org/Team%3ANanjing-China/Partnership)

[ACHIEVEMENTS](https://2020.igem.org/Team%3ANanjing-China/Achievements)

![picture display fail](https://static.igem.org/mediawiki/2020/6/68/T--Nanjing-China--cover.jpg  "cover picture")
Experiments

Preparation of polyP

Purpose:

To prepare a sufficient amount of pure polyP with suitable chain length and suitable for binding.

Experimental results:

1. Through bacterial synthesis, we obtained 839.28mg of polyP in the bacterial body.

2. After extraction, we obtained 489.89mg of pure polyP product.

3. Through the ethanol separation method, we finally obtained 214.67mg of polyP 45 with a suitable chain length that can be used in subsequent binding experiments.

4. We have obtained a set of methods that can efficiently produce polyP, and determined the yields of extraction and purification steps. The polyP plasmid is highly expressed in Citrobacter fujii. After being expanded in LB medium for 12 hours, it is inoculated into the wastewater medium. The bacteria can use the inorganic phosphorus in the synthetic wastewater to synthesize polyphosphate. The maximum yield is about 20mg/L.

5. We have clarified the interference factors and their effects in each link in the production of polyP, and thus determined the methods that can increase the yield of polyP. Our plasmid is resistant to kanamycin, so it grows predominantly in LB medium supplemented with kanamycin. At the same time, it is the only predominant species in wastewater medium due to the scarcity of nutrients, but in wastewater medium The number of reproduction is not much and more energy is used to synthesize polyphosphate, and the yield is the highest 14 hours after inoculation, and the prolonged time may lead to a decrease in yield and the decomposition of polyphosphate. Among them, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest system output and the highest conversion efficiency. Almost all inorganic phosphates can be converted into polyphosphates under the condition of no other bacteria pollution and normal bacterial viability.

6. The content of polyP synthesized by CPP recombinant engineering bacteria at different culture time.

![](https://static.igem.org/mediawiki/2020/0/0b/T--Nanjing-China--experiment_picture_1.jpeg)

Figure 1 The change of polyP synthesis in engineering bacteria over time

As shown in Figure 1, at the beginning of the culture, the relationship between the amount of polyP synthesis in CPP and the time is close to linear growth, and the polyP production reaches its maximum after 16 hours.

+36GFP preparation

Purpose:

To prepare high-purity, super charged GFP(+36GFP) that can be used in binding experiments.

Experimental results:

We ligated the +36GFP target gene sequence to the PET29a vector to obtain the recombinant plasmid PET29a-+36GFP. The recombinant plasmid was transferred to the competent cell BL21 (DE3), and the final concentration of 1mM IPTG was induced at 16°C overnight for subsequent use of protein purification. In the experiment of purifying +36GFP, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify +36GFP (to achieve the purification of positively charged protein). The SDS-PAGE detection results of the protein purified by the above two columns are shown in the figure below:

![](https://static.igem.org/mediawiki/2020/6/6a/T--Nanjing-China--experiment_picture_2.jpeg)

Figure 2 SDS-PAGE diagram of each solution during purification using ion exchange column.

![](https://static.igem.org/mediawiki/2020/3/3e/T--Nanjing-China--experiment_picture_3.jpeg)

Figure 3 SDS-PAGE detection results of +36GFP and spidroin after purification.

In the lane corresponding to the sample solution, a band near 30kDa appeared, and no band of 30kDa appeared in the effluent of the sample, indicating that +36GFP in the sample solution can bind to the ion exchange column. In the corresponding lane of Binding Buffer, there is no band of 30kDa, which means that +36GFP cannot be eluted by Binding Buffer, and the sample contains less contaminated protein. In the effluents of eluate 1 and eluate 2, obvious bands appeared near 30kDa, indicating that +36GFP was eluted and +36GFP was successfully purified. Combined with the electrophoresis results of +36GFP after purification in Figure 3, there is a thicker band near 30kDa, which further shows that +36GFP was successfully purified.

+36GFP-SP fusion protein preparation

Purpose:

To prepare large amounts of high-purity, super charged GFP(+36GFP) fused with SP that can be used in binding experiments.

Experimental results:

We attached +36GFP-SP fusion protein gene sequence to PET28a vector and obtained the recombinant plasmid PET28a-+36GFP-SP, which was transformed to the competent cell BL21 (DE3) and induced by 1M IPTG at 25℃ overnight for later protein purification. The protein purification result shows that +36GFP-SP fusion protein produced by recombinant plasmid PET28a-+36GFP-SP can interact with Ni column and led to the successful purification. The SDS-PAGE detection results of the protein purified by the two kinds of columns are shown in the figure below:

![](https://static.igem.org/mediawiki/2020/2/2e/T--Nanjing-China--engneering_picture_3.png)

Figure 3.1 Result of +36GFP-SP SDS-PAGE

In reference to figure 3.1, bands of 70kDa appeared in both total protein track and supernatant track, while no band of 70kDa appeared in the precipitation track. This indicates that +36GFP-SP fusion protein gene was successfully expressed by BL21 and most +36GFP-SP fusion protein existed in supernatant after cell disruption, which made it of value for the following purification step. Moreover, combined with the electrophoresis results of +36GFP-SP after purification in Figure 1, a weak band near 70kDa appeared, which further indicated the success of +36GFP-SP purification.

The combination experiment of +36GFP and polyP

Purpose:

To test whether the combination of polyP and +36GFP will increase the thermal stability, and to explore conditions for the subsequent binding of polyP and spidroin.

Experimental results:

We first determined the phase separation boundary where +36GFP binds to polyP, and selected the corresponding polyP and +36GFP concentrations in the phase separation boundary to perform binding experiments.The results of our experiments to find the boundary are shown in the figure3.1 below. The red dots show the binding condition of the two within the boundary. As can be seen from the fluorescence diagram on the left, the two can be combined into a tight substance at a specific concentration within the boundary.

![](https://static.igem.org/mediawiki/2020/e/e2/T--Nanjing-China--experiment_supplement2.jpg)

Figure 3.1 In vitro phase separation of +36GFP driven by polyP

PolyP (Sigma, Type45, Item S4379) with a final concentration of 3200μM and +36GFP (with an electrostatic charge ratio of 32) with a final concentration of 2.78μM are mixed in equal volumes, and the two are combined in the solution through electrostatic interaction to obtain a polyP-GFP solution. Next, we characterize the stability of the fluorescence of +36GFP before and after the polyP is combined with the degree of decay of the GFP fluorescence intensity with time before and after the combination, and the effect of different temperatures on the fluorescence intensity.

**1.GFP fluorescence intensity changes with time before and after binding at room temperature.**

![](https://static.igem.org/mediawiki/2020/f/f4/T--Nanjing-China--experiment_picture_4.jpeg)

Figure 4 Fluorescence of polyP-GFP, GFP and control solution after 3h, 7h, 24h, 48h combination

As shown in Figure 4, the fluorescence of GFP will decrease significantly with time. Under the same conditions, +36GFP combined with polyP has higher fluorescence brightness than +36GFP without polyP, especially after 48h, the fluorescence of +36GFP combined with polyP is stable and the fluorescence brightness is high, which shows that polyP can stably protect GFP, prolong the fluorescence time of GFP. Therefore, we can preliminarily speculate that polyP can protect the stability of GFP fluorescence.

Next, 5.56μM +36GFP and 10μM +36GFP were mixed with different concentrations of polyP in equal volumes, respectively, and the fluorescence intensity was detected after 0h, 3h, 24h, and 48h incubation.

![](https://static.igem.org/mediawiki/2020/9/91/T--Nanjing-China--experiment_picture_5.jpeg)

Figure 5 The effect of different polyP binding concentration on GFP fluorescence intensity (0~48h)

As shown in Figure 5, under the same binding time, as the binding concentration of polyP increases, the fluorescence stability of polyP increases, the fluorescence intensity increases significantly, and then slowly increases. When the binding concentration of polyP is low, GFP is excessive and polyP binds to part of GFP. At this time, increasing the binding concentration of polyP will increase the GFP binding to polyP and increase the fluorescence intensity.

All the above are the results we obtained from our in vitro binding experiments. We also tried binding GFP with polyP in vivo, and the results are shown in the figure5.1. The results showed that +36GFP and polyP could be synthesized simultaneously in vivo and directly combined in bacteria.These results can help us better apply the experimental results to the production of our products.

![](https://static.igem.org/mediawiki/2020/e/e8/T--Nanjing-China--experiment_picture_5.1.jpeg)

Figure 5.1 +36GFP binding to polyP in vivo

**2. Thermal stability of GFP fluorescence intensity before and after polyP binding.**

(1) PolyP (6400μM) and +36GFP (5.56μM) are mixed in equal volume, in the phase boundary of liquid-liquid separation, incubated at room temperature for 30min, and then 25, 30, 40, 50, 60, 70, 80, 90, 100 Incubate at ℃ for 10 min. After cooling, the fluorescence intensity is detected by the microplate reader.

![](https://static.igem.org/mediawiki/2020/a/a9/T--Nanjing-China--experiment_picture_6.jpeg)

Figure 6 The influence of temperature on the fluorescence intensity of GFP

After GFP is combined with polyP, the fluorescence intensity after incubation at different temperatures is greater than that of GFP that is not combined with polyP, indicating that the combination of polyP and GFP can protect GFP, and the relative thermal stability of GFP is increased. To further determine the influence of polyP on the thermal stability of +36GFP, we did the following experiment.

(2) The final concentration of polyP (0,100,400,800,1600,3200μM) and the final concentration of +36GFP (2.78μM) were mixed in equal volume, incubated at room temperature for 30min, and then 25,30,40,50,60,70,80,90,100℃ After incubating for 10 minutes, after cooling, the fluorescence intensity is detected by the microplate reader

![](https://static.igem.org/mediawiki/2020/d/d4/T--Nanjing-China--experiment_picture_7.jpeg)

Figure 7 The effect of different final concentrations of polyP on the fluorescence stability of +36GFP

As shown in Figure 7, with the increase of polyP binding concentration, the fluorescence intensity of +36GFP increases, and when the fluorescence intensity decays to 50%, the corresponding temperature Tm also rises significantly (Figure 8), indicating that the combination of polyP and +36GFP can increase+ The thermal stability of 36GFP.

![](https://static.igem.org/mediawiki/2020/7/79/T--Nanjing-China--experiment_picture_8.jpeg)

Figure 8 The effect of different concentrations of polyP on the Tm value of +36GFP

**3. The change of fluorescence intensity in protein denaturation solution before and after +36GFP combined with polyP.**

+36GFP+polyP: polyP (0,200,800,1600,3200,6400) and +36GFP (5.56μm) mixed in equal volume (50ul+50ul), respectively incubated at room temperature for 30min, then mixed with denaturing solution (100ul) and incubated for 30min, enzyme label The instrument detects the fluorescence intensity. (1X denaturing solution; 4M urea, 0.5% SDS, 3M guanidine hydrochloride. 2X: 8M urea, 1% SDS, 6M guanidine hydrochloride)

![](https://static.igem.org/mediawiki/2020/2/29/T--Nanjing-China--experiment_picture_10.jpeg)

Figure 9 The effect of different binding concentrations of polyP on the fluorescence intensity of +36GFP in denaturing solution

[PREPARATION OF POLYP](#cover_picture_div)

[#1](#cover_picture_div)

[+36GFP PROTEIN PREPARATION](#+36GFP PROTEIN PREPARATION)

[#2](#+36GFP PROTEIN PREPARATION)

[+36GFP-SP FUSION PROTEIN PREPARATION](#+36GFP-SP FUSION PROTEIN PREPARATION)

[#3](#+36GFP-SP FUSION PROTEIN PREPARATION)

[THE COMBINATION EXPERIMENT OF +36GFP AND POLYP](#THE COMBINATION EXPERIMENT OF +36GFP AND POLYP)

[#4](#THE COMBINATION EXPERIMENT OF +36GFP AND POLYP)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Nanjing-China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)