iGEM 2019 — Team Nanjing-China

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# Team:Nanjing-China/Notebook

Team:Nanjing-China

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* [Parts](https://2019.igem.org/Team%3ANanjing-China/Parts "Parts")
* [Hardware](https://2019.igem.org/Team%3ANanjing-China/Hardware)
* [Human Practices](https://2019.igem.org/Team%3ANanjing-China/Human_Practices)
* [Achievements](https://2019.igem.org/Team%3ANanjing-China/Achievements)

## Notebook

1. [Home](https://2019.igem.org/Team%3ANanjing-China)
2. Notebook

## March

We established Nanjing-China 2019 in March this year. Both the team leader and all team members are new iGEMers, in which case great challenges were ahead of us. We determined that we work on gas sensors and then read a lot of papers and conceived the outline of our project.

## April

In April, we started synthesizing primers and genes with the help of our PI weiwei and instructor Xin Wang. We also made a proposal within School of Life Science to secure funding and support.

## May to August

From May to August, we were busy with human practice and experiments. At the end of August, we sent 8 students to attend CCIC in Shenzhen and met up with iGEMers from all over China. We made a presentation about our project so that we got to know what they thought about our project.

## September and October

In September and October, we visited SEU in Nanjing to discuss our model, and we also formed collaboration with team Fudan-TSI. We also spread no effort in building wiki and modeling before November. We had been making beautiful original design throughout the whole time. We were so lucky to have talented and hard-working students to get everything neatly done before the Giant Jamboree.

# Protocol

AxyPrep Plasmid Miniprep Spin Protocol

```
1. Collect 1-4 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria.
Decant or pipette off as much of the supernatant as practical.
Note: When using rich broths such as LBG or 2×YT, reduce the culture volume by half. Excessive
 bacteria will reduce lysis efficiency, resulting in low yield and reduced purity of the plasmid DNA.
 Do not exceed 2 ml of bacterial culture grown in rich broth.
2. Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing. Please be sure that the
bacteria are completely resuspended before proceeding.
Note: Be sure that RNase A has been added into Buffer S1.
3. Add 250 μl of Buffer S2, and mix by gently inverting the tube for 4-6×. Do not vortex.
Note: Vigorous shaking or vortexing will cause shearing of the bacterial genomic DNA and result in the
 contamination of the plasmid DNA.
Note: After use, the buffer S2 bottle should be closed immediately in order to avoid neutralization of
NaOH by ambient CO2.
Note: Buffer S3 (Step 4, below) must be added within 5 minutes.
4. Add 350 µl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to
 clarify the lysate. Do not vortex.
Note: Vigorous shaking or vortexing will result in contamination with genomic DNA.
5. Place a Miniprep column into an uncapped 2 ml Microfuge tube (provided). Transfer the clarified
supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube
 to microcentrifuge and spin at 12,000×g for 1 minute.
6. Optional step: Buffer W1 Wash Washing with Buffer W1 is required only in cases where the plasmid has
 been propagated in an endA+ bacterial strain. These strains often exhibit high levels of endonuclease
 activity which will degrade the plasmid DNA.
Proceed to Step 7 if an endA- bacterial strain is used.
Pipette 500 µl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
7. Pipette 700 µl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
Note: Make sure that the volume of ethanol specified on the bottle label has been added to the Buffer
W2 concentrate
8. Optional Step: Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into
 the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column and centrifuge at 12,000×g for
 1 minute.
Note: Two washes with Buffer W2 are used to ensure the complete removal of salt, eliminating potential
problems in subsequent enzymatic reactions.
9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge
tube. Centrifuge at 12,000×g for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube (provided). To elute the purified
plasmid DNA, add 60~80 µl of Eluent (or deionized water) to the center of the membrane. Let it stand for
 1 min at room temperature. Centrifuge at 12,000×g for 1 minute..

```

Agarose Gel Electrophoresis

```
1.Pouring a Standard 1% Agarose Gel:
1.1Measure 1 g of agarose.
1.2Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
1.3 Microwave for 1-3 min until the agarose is completely dissolved (Microwave for 30-45 sec, stop and
 swirl, and then continue towards a boil)
1.4 Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on
the flask), about 5 mins.
1.5 Add dye to a final concentration of 1/10000.
1.6 Pour the agarose into a gel tray with the well comb in place.
1.7 let the gel sit at room temperature for 20-30 mins, until it has completely solidified.

2.Loading Samples and Running an Agarose Gel:
2.1 Add loading buffer to each of DNA samples.
2.2 Once solidified, place the agarose gel into the gel box (electrophoresis unit).
2.3 Fill gel box with 1xTAE until the gel is covered.
2.4 Carefully load a molecular weight ladder into the first lane of the gel.
2.5 Carefully load your samples into the additional wells of the gel.
2.6 Run the gel at 120 V until the dye line is approximately 75-80% of the way down the gel.A typical
run time is about 0.5 hours.
2.7 Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel
 from the gel box.
2.8 Using any device that has UV light, visualize DNA fragments.
```

PCR amplification

```
Takara PrimerSTAR Max DNA Polymerase
PCR Reaction solution composition（50 μl system）:
Volume            Final Conc.
2×PrimeSTAR GC Buffer (Mg2+ plus)          25 μl                1×
dNTP Mixture (2.5 mM each)                  4 μ            l 200 μM each
Primer 1                                10 - 15 pmol        0.2 - 0.3 μM
Primer 2                                10 - 15 pmol        0.2 - 0.3 μM
Template                                ＜200 ng
PrimeSTAR HS DNA Polymerase (2.5 U/μl)     0.5 μl             1.25 U/50 μl
Sterile water                             up to 50 μl
```

PCR clean-up

```
AxyPrep PCR clean-up kit:
1. Add 3 volumes of PCR-A to 1 volume of the PCR sample and mix.
2. Place a spin column in a provided collection tube. Apply the sample to the column and centrifuge for
1min.
3. Discard flow-through and place the column back into the same tube.
4. Add 0.7 ml Buffer W2 to the column and centrifuge for 30–60 s.
5. Discard flow-through and place the column back in the same tube.
6. Add 0.4 ml Buffer W2 to the column and centrifuge for 30–60 s.
7. Place the column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 30 µl water (pH 7.0–8.5) to the center of the membrane, let the column stand for 1
min, and centrifuge the column for 1 min.

```

Restriction enzyme digestion

```
System：10ul/50ul
10ul system：
DNA/plasmid：  8ul
Buffer：        1ul
Enzyme1：     0.5ul
Enzyme2：     0.5ul
Reaction temperature: 37℃
Reaction time was determined by enzyme activity and sample amount.
```

Ligation

```
10ul system:
Insert          calculate
Vector         calculate
T4 DNA ligase   0.5ul
10×buffer       1ul
Water         up to 10 μl
Control a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
Gently mix the reaction by pipetting up and down and microfuge briefly.
Incubate at 16°C for 30 minutes.
```

Transformation Protocol Using Heat Shock

```
1. Take competent E.coli cells from –80℃ freezer. Keep tubes on ice.
2. Turn on water bath to 42℃
3. Add 10ul of circular DNA into E.coli cells. Incubate on ice for 30 min.
4. Put tube(s) with DNA and E.coli into water bath at 42℃ for 80 seconds.
5. Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
6. Add 1 ml of SOC medium. Incubate tubes for 1 hour at 37℃ with shaking at 200rpm.
7. Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added – usually
 Ampicillin or Kanamycin.) Grow overnight.
8. Pick colonies about 12-16 hours later.

```

Competent DH5alpha cell preparation

```
Day1:
1. Flame the metal inoculating loop until it is red got and then cools it down.
2. Scrape off a portion from the top of the frozen glycerol stock.
3. Streak it onto the LB plate.
4. Put the stock back to -80℃ immediately.
5. Leave the plates for 5 minutes and place them upside down in the 37℃ incubator for 16-20 hours.
Day2
6. Pick a single colony into 5ml of LB medium.
7. inoculate the culture overnight at 37℃ with shaking at 200rpm.
Day3
8. Inoculate 100ml LB medium with 1ml of saturated overnight culture.
9. Shake at 37℃ until OD600=0.5 (usually 2-3 hours).
10. Place in an ice bath for 10 minutes.
11. Pre-cool solution, centrifuge, pipette tips, falcon, Eppendorf.
12. Transfer the culture into two pre-chilled 50ml tubes.
13. Centrifuge at 4000rpm for 10 minutes at 4℃.
14. Remove the medium, resuspend the cell pellet with 30ml ice-cold 100mM CaCl2-MgCl2 by swirling on ice
gently.
15. Incubate on ice for 10 minutes.
16. Centrifuge at 4000rpm for 10 minutes at 4℃.
17. Remove the medium, resuspend the cell pellet with 2ml ice-cold 100mM CaCl2- glycerol by swirling on
ice gently.
18. Freeze 100ul aliquots and store in -80℃.

```

Activate the strains

```
1. Flame the metal inoculating loop until it is red got and then cools it down.
2. Scrape off a portion from the top of the frozen glycerol stock.
3. Streak it onto the LB plate.
4. Put the stock back to -80℃ immediately.
5. Leave the plates for 5 minutes and place them upside down in the 37℃ incubator for 8-10 hours.
```

Cultivate in LB

```
1. Pick a single colony into 5ml/50ml of LB medium (with appropriate antibiotic added).
2. Inoculate the culture overnight at 37℃ or for 8-10 hours with shaking at 200rpm.
```

Cultivate in Synthetic municipal wastewater (SMW)

```
1. Inoculate the culture overnight at 37℃ or for 8-10 hours with shaking at 200rpm.
2. Calculate the culture beforehand to attain an initial OD600 of 0.15 in SMW.
3. Centrifuge at 6000rpm for 5 minutes, and wash once with deionized water.
4. Resuspend the cell pellet with 5ml SMW, transfer them all to the 100ml SMV, Incubating  for 10-14 hours at
 37℃ with shaking at 200rpm.
5. Monitor the OD600 and the pi concentration each hour, until the bacteria cannot grow anymore or cannot remove
 the pi anymore.

```

Media component

```
A.LB medium：
Tryptone           10g/L
Yeast extract        5 g/L
NaCl              10g/L
Deionized water    up to 1 L
* adjust pH： 1M  NaOH     1ml

B.Synthetic municipal wastewater (SMW)
Glucose           0.3g/L
Sodium acetate     0.15g/L
Tryptone          0.1g/L
Yeast extract       0.01g/L
K2HPO4 ·3H2O      0.1472g/L
MgSO4 ·7H2O      0.262g/L
NH4Cl             0.18g/L
NaCl              0.05g/L
```


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# Team:Nanjing-China/Experiments

Team:Nanjing-China

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## Experiments

1. [Home](https://2019.igem.org/Team%3ANanjing-China)
2. Experiments

In order to make phosphorus removal more effective, citrate frederik bacillus was selected instead of E.coli. Their different efficiency was due to the difference of the two bases at the position of ppk1 gene 327 and 328.

![](https://static.igem.org/mediawiki/2019/4/46/T--Nanjing-China--ex1.jpg)

E.coli

![](https://static.igem.org/mediawiki/2019/c/c1/T--Nanjing-China--ex2.jpg)

C. freundi

Through the experiment, we verified that the phosphorus removal effect of the difference is real， We transferred the ppk1 gene of C. freundi(CF) and E.coil(DH5α) into C. freundi and E.coil by different gene copy number strategies, eventually identified only CF with medium copy of the CF ppk1 genes can achieve the best effect of phosphorus.

![](https://static.igem.org/mediawiki/2019/5/5c/T--Nanjing-China--ex3.jpg)

p.s.:M stands for medium copy strategy, and H stands for high copy strategy

We constructed pBBR1MCS2-ppk1 using the genomic DNA of bacillus citrate frumenii (CWT) as a template. Firstly, ppk1 was amplified by PPKF and PPKR primers. After sequencing confirmation, PCR products were digested by Kpn I and Hind III and inserted into pBBR1MCS2 (pBR322 ori) to obtain pBBR1MCS2-ppk1, and then obtain CWT transformed from pBBR1MCS2-ppk1, the strain was named CPP, it is the core engineered bacterium of our experiment.

![](https://static.igem.org/mediawiki/2019/d/d5/T--Nanjing-China--ex4.jpg)

![](https://static.igem.org/mediawiki/2019/5/5e/T--Nanjing-China--ex5.jpg)

The main function of this strain is to enrich PO43- in sewage by synthesizing Poly P in the cell. The engineered bacteria can overexpress ppk1 and reversibly catalyze the transfer of the terminal phosphate (Pi) from ATP to the growing poly P chain. After that, the expression product of PPX can released PO43- to the outside of the cell under certain conditions, so that we get high concentration of Pi to precipitate struvite.

![](https://static.igem.org/mediawiki/2019/c/c2/T--Nanjing-China--ex6.jpg)

![](https://static.igem.org/mediawiki/2019/c/c9/T--Nanjing-China--ex7.jpg)

After getting the engineered bacteria, we designed the following three experimental parts: struvite formation, design device, and plant experiment. In the first part of the experiment, we explored the conditions of struvite precipitation in advance and chose the conditions with the highest phosphorus removal rate.

In the second part of the experiment, we designed a device that contains five modules. The first module is filled with synthetic wastewater containing phosphorus. Then, the wastewater can be drawn into the second module to supply the nutrition of engineered bacteria, which could effectively remove phosphorous from the waste water. In addition, the second module includes aeration devices and the MBR biofilm reactor. The phosphorus-free water treated by the engineered bacteria will pass through the MBR membrane bioreactor and enter the third module, while the bacteria, trapped by the membrane, will continue to remove phosphorus in the second module. We use a small aeration device to supply oxygen and a large aeration device to avoid bacterial blockage on the membrane. After the engineered bacteria have absorbed enough phosphorus, they will be pumped to the fourth module to release phosphorus; thus, we can get a high concentration of phosphorous containing solution. It will be mixed with the high ammonia nitrogen wastewater containing magnesium ions and ammonium ions in proportion in the fifth module to generate struvite precipitation.

In the third part of the experiment, we used the obtained struvite as fertilizer to grow soybean and lettuce, and make comparison with the full-fertilizer group and the phosphor-free group to explore the utility of struvite as fertilizer.

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# Team:Nanjing-China/Results

Team:Nanjing-China

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  + [Model](https://2019.igem.org/Team%3ANanjing-China/Model)
  + [Experiments](https://2019.igem.org/Team%3ANanjing-China/Experiments)
  + [Demonstrate](https://2019.igem.org/Team%3ANanjing-China/Demonstrate)
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* [Human Practices](https://2019.igem.org/Team%3ANanjing-China/Human_Practices)
* [Achievements](https://2019.igem.org/Team%3ANanjing-China/Achievements)

[The best bacteria of Pi removal #1](#aa)

[Expression of ppk/ppx #2](#bb)

[MAP precipitation #3](#cc)

[Device #4](#dd)

[Botanical experiments #5](#ee)

## Results

1. [Home](https://2019.igem.org/Team%3ANanjing-China)
2. Results

# 1、The best bacteria of Pi removal: CPP

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 naturally 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. Benefiting from this difference,C.freundii could achieve a dramatic increase of intracellular polyP accumulation.

As shown in Figure 1a, it is obvious that bacteria with different plasmids have disparate behavior in the SMW. Both CWT and DH5alpha served as controls. Associated with the synthesis of PolyP was the continuous uptake of exogenous Pi from the SMW, and for CPP, a maximal Pi removal of 19 mg of P/L was then achieved at 14 h. However,the smaller amount of Pi removed in the other three strains could be attributed to their need for growth, which formed a background TP content.

![](https://static.igem.org/mediawiki/2019/1/1a/T--Nanjing-China--results1.jpg)

Figure 1a

As mentioned above, the synthesis of PolyP by ppk1 requires ATP, which suggests that, synthesizing excessive PolyP may affect the growth of bacteria. We tested the influence of different-copies plasmid strategy in the E.coli and CF for the phosphorus removal(figure1b), as M stands for medium copy strategy, and H stands for high copy strategy. As a result, Citrobacter freundii with medium copy CFPPK can remove most phosphorus with least COD consumption.An approximately 60% decrease in Pi removal of CF-HCPP relative to that of CF-MCPP suggested that the cooccurrence of a second high-copy plasmid impacted the genetically engineered enhancement of polyP biosynthesis.

![](https://static.igem.org/mediawiki/2019/6/61/T--Nanjing-China--results2.jpg)

Figure 1b

However, polyP degradation accompanied by Pi secretion occurred in both CPP and CWT between 15 and 16 h. Since then, they entered the stage of Pi release until their intracellular polyP pools were totally depleted at 92 h (Figure 1d). This result implied that we can recycle phosphorus from CPP in the form of Pi, which will be discussed below. We detected OD600 both in two stages, and it was obvious that CF, that had higher biomass, was more suitable for SMW than CWT(Figure1c).

![](https://static.igem.org/mediawiki/2019/9/93/T--Nanjing-China--results3.jpg)

Figure 1c

![](https://static.igem.org/mediawiki/2019/6/6a/T--Nanjing-China--results4.jpg)

Figure 1d

At this point, we asked whether the polyP content of CPP could be further increased. The intracellular polyP of heterotrophic bacteria is essentially derived from exogenous Pi and the carbon source (represented as COD). In the transition phase of intracellular polyP synthesis and degradation, a certain amount of Pi (11 mg of P/L) remained in the supernatant, whereas COD was almost depleted (Figure 1b and Figure S1).

Therefore, a lack of available COD, not Pi, likely accounted for further accumulation of intracellular polyP in CPP. To directly test this idea and avoid growth dilution effects, filtersterilized glucose was added to a final concentration of 10 mg of COD/L (a value calculated on the basis of Figure S1) per hour. Nevertheless, the polyP content of CPP was not further increased, and Pi release continued (data not shown).

# 2、Expression of ppk/ppx

In order to prove that it is the overexpression of ppk1 gene that enables CPP high efficiency in Pi removal, we did qRT-PCR analysis to figure out the expression of ppk1 gene and ppx gene over time. As shown in the figures, we take 16S gene, a housekeeping gene, as control and set as 1, the extra expression of ppk1 at different times in CPP was decided by the ppk1 on the plasmid(Figure 2a), and the expression of ppx remained stable and low(figure2b), that is why the Pi secretion occurred in 15-16 hours later.

![](https://static.igem.org/mediawiki/2019/4/41/T--Nanjing-China--results5.jpg)

Figure 2a

![](https://static.igem.org/mediawiki/2019/8/89/T--Nanjing-China--results6.jpg)

Figure 2b

# 3、MAP precipitation

Taking highest phosphorus removal rate as the standard, we did a series of experiments to figure out the best conditions for struvite precipitation. Associated papers have demonstrated that the best pH for precipitation is 9, and molar ratios of N, Mg and P are also important. In contrast, temperature and reaction time are relatively of less vitality, so we chose pH=9 and start to find out the best Molar ratios of N, Mg and P. According to the Figure3a, when N and Mg were of slightly larger molar ratios than P, the deposition rate of struvite was greatly increased. Considering the cost, we chose pH=9, and molar of N:Mg:P= 1.25:1.25:1 as the best reaction options, under this condition, the Pi removal rate in the SMW could achieve 98.14%(Figure3a). The precipitation (Figure3b) was proved to be MAP via EDS, SEM and XRD, the electron microscopy images and the contrast are presented. (figure3c,d).

![](https://static.igem.org/mediawiki/2019/e/e9/T--Nanjing-China--results7.jpg)

Figure 3a

![](https://static.igem.org/mediawiki/2019/c/c8/T--Nanjing-China--results8.jpg)

Figure 3b

![](https://static.igem.org/mediawiki/2019/c/c0/T--Nanjing-China--home2.jpg)

Figure 3c

![](https://static.igem.org/mediawiki/2019/9/9b/T--Nanjing-China--results10.jpg)

Figure 3d

# 4、Device

![](https://static.igem.org/mediawiki/2019/7/75/T--Nanjing-China--results11.jpg)

Figure 4a

![](https://static.igem.org/mediawiki/2019/8/89/T--Nanjing-China--results12.jpg)

Figure 4b

![](https://static.igem.org/mediawiki/2019/c/cd/T--Nanjing-China--results13.jpg)

Figure 4c

As the most important part of this study, we sought to leverage the strengths of CPP to remove and concentrate Pi from SMW. Consistent with the results above the in-reactor Pi was completely depleted within 4 h. Thereafter, practically Pi-free water was consistently obtained through a continuous withdrawal of the reactor supernatant until 0.02 mg Pi/L was detected in the permeate stream at 16 h. During this period, Pi from 85 L SMW was fully taken by CPP and mainly converted to a concentrated form (i.e., intracellular polyP). After membrane concentration, 9 L of an enriched CPP cell suspension was obtained and then subjected to Pi release. At the end of the Pi release phase, a cell suspension with supernatant Pi concentrations of ≤62 mg Pi/L was formed; namely, Pi was effectively enriched up to 8-fold.

As confirmed by a recovery efficiency calculation, almost all Pi was successfully concentrated, apart from the portion assimilated by CPP. These results suggest that full-scale Pi removal and enrichment using CPP are technologically feasible.

# 5、Botanical experiments

In order to verify the fertilizer properties of struvite, we designed a positive control group of complete fertilizer, the negative control group without phosphorus and the experimental group with struvite. The complete fertilizer group used market compound fertilizer, the struvite group took struvite as the source of phosphate fertilizer, and the phosphorus free group used fertilizer without phosphorus element.Having been applied the same amount of fertilizer, the growth of struvite group was the same as that of the whole fertilizer group, but the growth without phosphorus was a little worse. However, the total production of struvite group was the highest, possibly because the low solubility of struvite makes it a high-quality slow-release fertilizer.

![](https://static.igem.org/mediawiki/2019/7/75/T--Nanjing-China--results14.jpg)

Figure 5a

![](https://static.igem.org/mediawiki/2019/7/70/T--Nanjing-China--results15.jpg)

Figure 5b

![](https://static.igem.org/mediawiki/2019/b/b3/T--Nanjing-China--results16.jpg)

Figure 5c