iGEM 2021 — Team NDNF_China

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# Team:NDNF China/Notebook

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## Week 1 (6.24-6.30)

![](https://static.igem.org/mediawiki/2021/0/0b/T--NDNF_China--iconfv1.png)

The chromoprotein circuit induced by arabinose, the L-dopa expression circuit induced by caffeine and the primers needed for the construction of gene circuit were preliminarily designed, and the primers needed for the construction of mercury ion detection circuit were sent to the company for synthesis.

We also purchased reagents needed for our experiments.

## Week 2 (7.1-7.7)

![](https://static.igem.org/mediawiki/2021/1/13/T--NDNF_China--iconfv2.png)

Part one

PCR amplification plasmid backbone and corresponding parts

PCR amplification for RGP plasmid, PSC101 plasmid and pET28a plasmid backbone. However RGP plasmid and PSC101 plasmid were not successfully amplified.

PCR amplification of the rest of the gene circuits: Four chromoprotein genes (gfasPurple, fwYellow, amiICP, spisPink), green and red fluorescent protein genes (sfGFP, mScarlet), mercury ion detection gene elements (chuA,HrtR,pHrt), Caffeine induced Circuit Element (Pbad-PROMOTER, IBMC378-PARTB, HpaB, HpaC)

Part two

First attempt to construct hydrogel core

First attempt to construct hydrogel core: the alginate solution containing bacterial strains was first dripped into the CaCl2 solution to prepare the core. However we found that the core prepared by this method was small, and a better way to prepare the core was needed.

## Week 3 (7.8-7.14)

![](https://static.igem.org/mediawiki/2021/1/17/T--NDNF_China--iconfv3.png)

Part one

The chromoprotein expression strain induced by arabinose was successfully constructed

Four chromoprotein circuits induced by arabinose were constructed by Gibson, and sequencing proved that all the circuits were successfully construct. Then the plasmids were preserved in DH5a and transferred into top10 strains to verify the normal functioning of gene circuits. Also, psc101 and RGP plasmid backbone were amplified again.

Part two

Successful construction of a proper sized hydrogel core

We prepared hydrogel cores of appropriate size by dripping alginate solution mixed with bacterial liquid onto parafilm.

However, in the process of preparing the shell, we found that directly using Acr-Bis solution to dissolve alginate could easily cause solidification in the process of dissolution, so it was necessary to change the dissolution method.

## Week 4 (7.15-7.21)

![](https://static.igem.org/mediawiki/2021/7/7a/T--NDNF_China--iconfv4.png)

Part one

Barcode design complete

Gibson assembly was used in building the mercury ion sensing plasmid and caffeine detection plasmid system that initially used red fluorescent protein (RFP) as an output reporter gene to assist us in identifying whether construction was correct. The construction was later validated by sequencing. Then the plasmids were kept in DH5a strains.

The construction of the caffeine detection plasmid failed. Showed by the sequencing results, some components were not connected to the plasmid and needed to be rebuilt.

Design barcode and send it to company for synthesis.

Part two

The preparation process of shell material was optimized

Through experiments, we finally chose to prepare Hidro shell by fully mixing 10% alginate with ACR-BIS solution in a certain proportion, so that the gel would not solidify in the process of dissolution.

In the process of soaking MES crosslinking system, we found that the shell was loose and easily fell off after soaking for 3h, so it could not form a flexible shell. Therefore, a better method was needed.

## Week 5 (7.22-7.28)

![](https://static.igem.org/mediawiki/2021/a/a9/T--NDNF_China--iconfv5.png)

Part one

Successfully constructing Hg detection plasmid

Gibson assembly was used to construct the caffeine detection plasmid, which was verified by plasmid sequencing, and then stored in DH5a strains.

The plasmid containing mercury ion detection circuit was transformed into top10 strains for functional test.

PCR amplified the Ptarget plasmid skeleton, barcode, arabinose inducer promoter and toxic protein Doc gene sequences for the future plasmid construction.

Part two

The ingredient formula of Hidro was further optimized

After consulting a large number of literatures and communicating with the author of the original lecture, we added calcium ions to MES crosslinker, and the experimental results show that adding calcium ions can help Hideo to form a tough and not easily fall off shell.

## Week 6 (7.29-8.4)

![](https://static.igem.org/mediawiki/2021/a/a7/T--NDNF_China--iconfv6.png)

Part one

First attempt to construct Gene editing plasmid

The Ptarget plasmid which was responsible for later incorporation of the barcode, arabinose induced toxic protein expression system into the genome was constructed by Gibson. However, through sequencing results, it was found that the Ptarget plasmid we used was a high copy plasmid, so the DOC protein gene sequence could not be added into the Ptarget plasmid. Therefore, we considered replacing the medium copy plasmid, and at the same time replacing the toxic protein gene.

Part two

Hidro that could achieve biocontainment was constructed

We tested the escape prevention function of our prepared Hidro. Results proved that the hidro still had the ability to prevent the leakage of internal microorganisms after 72 hours incubation in LB medium. However, at the same time, we found that some internal microorganisms in Hidro were unable to survive. Therefore, we want to improve the survival rate of internal microorganisms by reducing the time and measurement of microbial contact with toxic substances in the preparation process.

## Week 7 (8.5-6.11)

![](https://static.igem.org/mediawiki/2021/0/0b/T--NDNF_China--iconfv1.png)

Part one

The functional verification of mercury ion detection strain was successful

We reselected the toxin protein gene, constructed the Ptarget plasmid, and edited the top10 strains. The sequencing results showed that we successfully edited the genomes of top10 strains.

We tested the function of the previously constructed strain capable of sensing mercury ions in the environment and expressing fluorescent proteins, and the result shows it could work normally in the liquid medium environment.

Part two

Once again optimize the ingredient formula

We improved survival rate of enclosed microbes by making adjustments to shell component percentages, including lowering toxic reagent dosage of acrylamide, increasing dosage of TEMED which promotes crosslinking, reduce the time soaking in toxic MES crosslinking agent, etc. After that, we compared the new formula with the old one, and we found out that the survival ratio of microorganisms in the revised formula was significantly improved while the microbial escape rate was still considerably low.

## Week 8 (8.12-8.18)

![](https://static.igem.org/mediawiki/2021/1/13/T--NDNF_China--iconfv2.png)

Part one

The successful functioning of toxic protein was detected, and the caffeine-induced fluorescence producing strain was constructed successfully.

We sent the edited strains to the lab of GBSZ team, where they used Cas12 technology to test the barcode that we edited into the genome.

We tested the function of the toxic protein: Kid production induced by arabinose, and found that the system could function well, resulting in the death of bacteria.

Also, We transformed the caffeine detection plasmid and the caffeine response plasmid with red fluorescent protein as reporter gene into top10 strains, and tested its' function in LB culture. The experimental results proved that the circuit could work successfully as we predicted.

Part two

The arabinose induced color protein circuit and mercury ion detection circuit were proved to be capable of successfully working in Hidro.

We enclosed strains of arabinose induced chromoprotein expressive circuits and mercury ion detection circuit into Hidro for detection, and the results showed that these two strains could successfully function in Hidro.

## Week 9 (8.19-8.25)

![](https://static.igem.org/mediawiki/2021/1/17/T--NDNF_China--iconfv3.png)

Part one

Barcode detection success

Our barcode was successfully detected.

We replaced the reporter gene on the caffeine responsive plasmid with L-dopa expression gene, and verified the successful construction of the plasmid by sequencing.

Part two

Strains that sense caffeine molecules and express red fluorescent proteins were proved to normally work in HIDRO.

We enclosed the strains capable of sensing caffeine molecules and expressing red fluorescent protein into Hidro for functional testing, and the results showed that the strain could work normally and expressed fluorescent in Hidro.

## Week 10 (8.26-9.1)

![](https://static.igem.org/mediawiki/2021/7/7a/T--NDNF_China--iconfv4.png)

Part one

The caffeine-induced L-dopa expressive strain was successfully constructed

We tested the function of the caffeine-sensitive strain expressing L-dopa in LB medium, and the results proved that the strain could function successfully.

Part two

Various kinds of stress resistance of HIDRO were tested

We tested the protective effect of Hidro on internal microorganisms in a variety range of adverse environments, and the result founds that within the range of acidic pH=1.5, alkaline pH=10, at the temperature of 90℃ and drought for 72h, Hidro could provide protection to internal microorganisms in comparson than when directly exposed in liquid medium.

## Week11 (9.2-9.8)

![](https://static.igem.org/mediawiki/2021/a/a9/T--NDNF_China--iconfv5.png)

The caffeine-induced dopa production circuit and the food spoilage detection circuit were transferred into Hidro for functional verification.

We enclosed the strain that could sense caffeine molecules and express L-dopa into Hidro for functional testing, and the results showed that the strain could work normally in Hidro and secrete L-dopa to external environments.

![](https://static.igem.org/mediawiki/2021/d/d8/T--NDNF_China--footerff.png)

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# Team:NDNF China/Engineering

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## DBTL for Hidro material

![](https://static.igem.org/mediawiki/2021/0/0b/T--NDNF_China--iconfv1.png)

Overview: one of the major goals of NDNF\_China was to create a platform that could support functioning of bacterial strains while effectively preventing their leakage. Yet during testing of the initial design, quite a few setbacks arose. We subsequently made adjustments to our methods and eventually came up with hydrogel systems that met our objective and even exceeded some of our expectations.

### Design

The initial design of the hydrogel system included two layers, namely the inner alginate core and the tough outer shell.

The alginate core contains nutrients for bacterial growth and survival, and also provides an environment that supports their function, whilst the tough hydrogel coating with a limited pore size (5~50 nanometres) is permeable to small molecules, and highly resilient to outer pressure.

The outer shell combines a polyacrylamide network with an alginate network using covalent crosslink. During stretching, the former serves as a stretchy basis, while the latter unzips the ionic bonds between its chains.

### Build

Manufacturing the alginate cores and the shell.

The bead was made according to methods used in the original literature[1].

### Test

During our actual process of hygrogel making, not all intended functions materialised as anticipated:

#### Toughness

Using the original preparation method in literature, the tensile strength of the outer coating was far from meeting our expectations.

Its brittleness meant that once coated, it would become easily detached and could be peeled from the core without much effort.

[](https://static.igem.org/mediawiki/2021/1/1a/T--NDNF_China--esv1.mp4)

Video 1：demonstration of the comparative plasticity of the original and refined shell formulations

\*Note: We are well aware that it is prominent to wear gloves at all times. In explanation to why none were worn in this particular video, please refer to our [safety webpage.](https://2021.igem.org/Team%3ANDNF_China/Safety)

#### Biocontainment efficiency

Another problem we encountered was the inability of the shell to enclose strains. Leakage of the strains would occur as if there were no external layer.

![](https://static.igem.org/mediawiki/2021/b/b8/T--NDNF_China--esf1.png)

Figure 1: High escape frequency of the original shell formulation.

#### Manufacturing capacity

With reference to the original literature, we dribbled a pre-gel solution of the shell onto parafilm, where they could be subsequently coated onto the core by rolling with a pair of 200ul pipette heads. Yet only a single bead could be made per batch, which was an extreme waste of time. We were deeply set back by the tedious process and later came to a conclusion that this was going to be a large limiting factor on the number of hydrogels that could be produced per day.

#### Toxicity of outer shell

Later, when we were reviewing the literature article, we came over an experiment displaying toxicity effect of the crosslinking solutions on cell viability, and took this into consideration.

After discovering that a major component of the hydrogel --- acrylamide was also highly toxic, and could seriously damage survival of the strains, we further realised the solemnness of the situation.

Especially for a lot of engineered strains that are able to carry out their functions better at denser bacterial populations, such as the one we came over during our [partnership activities during human practice,](https://2021.igem.org/Team%3ANDNF_China/Partnership) this was a problem that demanded an urgent solution.

### Learning and Optimization

Taking all of the above into consideration, we set foot on a trail to optimize current methods, including making adjustments to the preparation process and the formulation of the hydrogel shell. Through [exchanging information with authors](https://2021.igem.org/Team%3ANDNF_China/Human_Practices) of the original literature, continuing to research more information from other articles, and some creative thinking, we were finally able to use results from our tests to optimize production of the hydrogel system.

#### Properties of the shell

From previous results, we discovered there still existed a huge room of improvement for toughness of the shell. After contacting the original authors via email, we discovered that we omitted a necessary component from the crosslinking solution--- calcium ions, which are responsible for ionically crosslinking the alginate chains. After more research from diverse articles[2-3], we included calcium ions into our formulation for the MES crosslinking solution. This successfully resulted in producing shells of desired toughness.

[](https://static.igem.org/mediawiki/2021/f/f1/T--NDNF_China--esv2.mp4)

video2: inclusion of calcium ions into the crosslinking solution produced shells of desired tensile strength

\*Note: We are well aware that it is prominent to wear gloves at all times. In explanation to why none were worn in this particular video, please refer to our [safety webpage.](https://2021.igem.org/Team%3ANDNF_China/Safety)

#### Bioontainment efficiency

Consulting the authors on our previous failure in enclosing strains, as well as extending our research to other sources revealed three factors that were the determinants of biocontainment efficiency.

1. Whether there were bubbles in the shell:

Degassing the mixture thoroughly was crucial towards preventing formation of air bubbles. Yet the extent of gas removal that was needed could hardly be achieved by equipment we previously used, leaving us with mixtures that were incompletely degassed. Another problem with using vacuum degassers was that the process took a long time and was inefficient. Although a vacuum degasser that could meet the stringent requirements was recommended by the authors, it was inaccessible, as purchasing equipment of that standard was not possible. Eventually, we turned our attention towards ultrasonic degassing as an alternative, as it was efficient to operate and less demanding on the equipment itself, being able to degas the pre-gel mixture within seconds. We were thus able to thoroughly degas the pre-gel mixture at a relatively fast speed.

2. Whether the shell had been evenly coated

During manual coating, the hydrogel core could be susceptible to being unevenly covered. In areas where coating was thin or had no coating, leakage of strains would occur.

3. The thickness of the outer shell layer

The authors highly suggested that we increase the layers of coating to make it thicker so that even coating could be ensured, and thus could effectively prevent leakage of the strains.

\*Note：The latter two factors were resolved alongside our optimization of the preparation process which will be introduced in the section below.

#### Preparation process

Previous trials in making hydrogels turned out to be time and effort demanding, as a lot of effort was needed in maintaining an even coating and spherical shape of the bead. Yet, this largely restricted the possibility of Hidro to achieve intended functions. We started to look for methods that were less labourious but at the same time did not incur too much cost.

Techniques applied in cooking spurred our thinking: We borrowed a method commonly used in mousse making--- sealing centres into the mousse mixture. After getting hold of appropriately sized moulds, we imitated the process by preloading the mould with mixtures of the outer shell using syringes, then carefully placing and centering the cores with tweezers. This successfully produced evenly coated hydrogels with suitable shell-thickness, significantly improving on both the quality of beads made and speed of the process. Adjusting the size of cores and moulds provided means for us to further control thickness of the shell.

For details of this process please visit protocols.

[Protocols >](https://2021.igem.org/Team%3ANDNF_China/Experiments)

#### Toxicity considerations

On increasing survival rate of enclosed strains, we reduced the soaking time of beads in the crosslinking solution from 3 hours to around an hour.

After further research, we also decided to decrease the ratio of acrylamide to N-methylenebiscrylamide. This encouraged crosslinking of polyacrylamide as well as increasing the rate of reaction, which reduced contact time of microbes with the toxic acrylamide and therefore considerably lowered toxicity effects on microbes enclosed within the core. Please visit our proof of concept webpage for details.

[Proof of Concept >](https://2021.igem.org/Team%3ANDNF_China/Proof_Of_Concept)

### Engineering Success

In the end, after a full engineering cycle, we succeeded in producing hydrogel systems that could prevent escape of strains, had low toxicity and could be manufactured in batch at high efficiency.

The hydrogels eventually produced had tough and tensile shells and ensured low escape frequency (Figure 2). Meanwhile, the improved shell formulation was less harmful towards the strains enclosed within.

![](https://static.igem.org/mediawiki/2021/6/66/T--NDNF_China--pocf20.png)

Figure 2: Hydrogels that could effectively contain strains were produced.

![](https://static.igem.org/mediawiki/2021/5/50/T--NDNF_China--esf3.png)

Table 1: Optimized composition of the components used to make the outer shell

## DBTL for Kill Switch

![](https://static.igem.org/mediawiki/2021/1/13/T--NDNF_China--iconfv2.png)

In summary: a key property of the Hidro system is the ability to achieve 'passive suicide': once escape is confirmed by CRISPR-Cas12a, staff can actively spray the inducer to induce microbes to produce toxic proteins to achieve suicide. After we completed the initial version of the design, we found many problems during testing. We then optimized the toxin selection and gene expression level of the kill switch system to finally achieve our goal.

### Design

In the initial design, the main parts of the kill switch are: 1. a user-customizable inducible promoter, such as arabinose promoter; and 2. a toxin gene.

The kill switch will eventually be integrated into the genome using the CRISPR-Cas9 system, thus maintaining the stability of the suicide genetic circuit. The genetic circuits design is shown in the following diagram.

![](https://static.igem.org/mediawiki/2021/8/88/T--NDNF_China--esf4.png)

Figure 3: The design scheme of the kill switch in Hidro system.

### Build

1. Assemble pTarget plasmids with gRNA, homologous arms, and kill switches using the Gibson assembly method. (This plasmid also contains Barcode sequences; for more information, please visit Proof Of Concept).

Genome integration site：E. Coli Top10 fimA coding region

gRNA sequence：ACCCGTTGCTGTTGTTGTAT

[Proof of Concept >](https://2021.igem.org/Team%3ANDNF_China/Proof_Of_Concept)

### Test

In our testing, we found the following problem: we found a large number of mutations in the translation start region of the toxin gene in the sequencing results, which led to the failure of the killing effect of the toxin.

![](https://static.igem.org/mediawiki/2021/c/cf/T--NDNF_China--esf5.png)

Figure 4: Mutations were found in the translation start region of the toxin gene.

### Learning and Optimization

Based on the results obtained in the test, we realized that the leakage of toxic protein expression caused by high-copy plasmids or arabinose promoter can cause bacterial growth restriction or death, thus affecting the effectiveness of plasmid assembly and genome engineering.

To find the appropriate expression strategies of the toxic protein, the following optimization design was carried out.

1. Changing plasmids with different copy numbers: the expression of leaked toxic proteins is reduced by tuning the plasmid copy number. We chose to convert the replication origin from the original CoE1 (several hundred copies) to p15A with only about 20-50 copies.

2. Using RBS Calculator（<https://salislab.net/software/>）to tune the translation level of toxic proteins: we calculte the original RBS intensity and designed RBS sequences with lower intensity using RBS Calculator developed by Sallis Lab.

Step 1: Determine the strength of the original RBS

![](https://static.igem.org/mediawiki/2021/d/d0/T--NDNF_China--esf6.png)

Step 2: Design RBS sequences with different intensities using RBS Calculator

![](https://static.igem.org/mediawiki/2021/b/b2/T--NDNF_China--esf7.png)

3. Different toxin genes with different killing mechanisms were chosen to find suitable candidates : Doc, Colicin and Kid were selected and tested for their killing effect in E. coli.

### Engineering Success

After changing the plasmids and the toxin gene, we found that the Kid toxin protein expressed on the mid-copy plasmid was successfully integrated into the genome and could achieve effective killing of bacteria.

![](https://static.igem.org/mediawiki/2021/9/94/T--NDNF_China--esf8.png)

Figure 5: The viability of E. coli with the Kid kill switch with or withour L-arabinose inducer.

## Reference

![](https://static.igem.org/mediawiki/2021/1/17/T--NDNF_China--iconfv3.png)

1. Sun, J.-Y. et al. Highly stretchable and tough hydrogels. Nature 489, 133–136 (2012).

2. Tang, T.-C. et al. Hydrogel-based biocontainment of bacteria for continuous sensing and computation. Nat Chem Biol 1–8 (2021) doi:10.1038/s41589-021-00779-6.

3. Darnell, M. C. et al. Performance and biocompatibility of extremely tough alginate/polyacrylamide hydrogels. Biomaterials 34, 8042–8048 (2013).

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# Team:NDNF China/Experiments

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* [HOME](https://2021.igem.org/Team%3ANDNF_China)
* PROJECT
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  + [Modelling](https://2021.igem.org/Team%3ANDNF_China/Model)
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* [PARTS](https://2021.igem.org/Team%3ANDNF_China/Parts)
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[](https://static.igem.org/mediawiki/2021/2/21/T--NDNF_China--Protocols.mp4)

## Plasmid Construction

![](https://static.igem.org/mediawiki/2021/0/0b/T--NDNF_China--iconfv1.png)
![](https://static.igem.org/mediawiki/2021/a/a3/T--NDNF_China--Plasmid_Construction.jpg)

## Hydrogel Preparation

![](https://static.igem.org/mediawiki/2021/1/13/T--NDNF_China--iconfv2.png)

[](https://static.igem.org/mediawiki/2021/d/db/T--NDNF_China--hydrogel_preparation_video2.mp4)

1. Sterilize 5% sodium alginate at 115°C for 15min, then mix with the bacteria liquid in a volume ratio of 2:1.

2. Form the core using syringes then soak in 5% calcium chloride solution.

3. Rinse the core and set aside to dry off before coating starts.

4. Sterilize 10% sodium alginate at 115℃ for 15min, mix with Acr-Bis solution of different concentration at ratio 1:4 (0.046% ammonium persulfate, 2000:1 tetramethylethylenediamine (TEMED) were added to the Acr-Bis solution in advance)

5. Preload the alginate and Acr-bis mixture into a mould, and lodge in the previously made core.

6. Soak in MES + Crosslinkers

a. Cross-linking system: 0.3M calcium chloride, 0.00125% of 1-ethyl-(3-dimethylaminopropyl) carbodiimide Amine (EDC), 0.000375% N-hydroxysuccinimide, and 0.00075% adipic acid dihydrazide (ADH) mixed, dissolved in MES buffer

b. MES buffer: 2-N-morpholino-ethanesulfonic acid MES 9.76g and NaCl 14.61g are mixed and dissolved, and the volume is adjusted to 500ml PH6.0

## Tests

![](https://static.igem.org/mediawiki/2021/1/17/T--NDNF_China--iconfv3.png)

### Escape prevention

1. Add 2 ml of LB medium plus antibiotics and CaCl2 (20mM) to each well of a 24-well plate.

2. Put the prepared hydrogel beads into the 24-well plate (one bead in each well).

3. Take 150 μL of medium and take photos at regular intervals. Use the absorbance OD600 for detection, then plot the escape curve.

### Colour expression

1. Encapsulate bacteria containing certain genetic circuit into hydrogel beads (procedures are mentioned above).

2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).

3. Beads containing engineered bacteria are incubated in LB plus antibiotics and arabinose at 37 °C.

4. Observe the color of each bead.

### Mercury sensing

**[bacteria]**

1. Cells are grown overnight at first, and are transformed into M9 (150 μL, 96-well plate) plus antibiotics and mercury ion (10μM) at the ratio of 1:100. Use the same amount of mercury-sensing bacteria and M9 medium plus antibiotics for serial dilution.

2. 8 h of incubation at 37 °C

3. Fluorecence tests using plate reader with the range of wavelength 485/535.

4. Bacteria liquid concentration tests using plate reader with the wavelength of 600.

5. Plot the fluorescence testing curve.

**[in beads]**

1. Encapsulate bacteria containing mercury sensing genetic circuit into hydrogel beads.

2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h.. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).

3. Beads containing engineered bacteria are incubated in LB plus antibiotics and mercury ion (10μM) at 37 °C for 8 h.

4. Test results can be analysed by fluorescence microplate assays or fluorescence microscope.

a. Fluorescence microplate assays:

i. Remove the shell of hydrogel beads using a razor blade and a tweezer.

ii. Place the hydrogel beads in a vitreous bead beater, add 0.5 mL of filtered, sterilized PBS. Homogenize hydrogel beads using bead beater.

iii. After centrifugation at 300g for 5min, 150 µL of supernatant was taken into 96-well plates, and microplate reader is used to test fluorescence in the wavelength range of 485/535 nm.

b. Fluorescence microscope: Slice the bead with a sharp razor blade at thickness of roughly 0.5 mm. The sliced sample was then imaged with a fluorescence microscope with excitation wavelength at 485 nm and emission wavelength at 535 nm.

### Caffeine sensing

**[bacteria]**

1. Cells are grown overnight at first, and are transformed into M9 (150 μL, 96-well plate) plus antibiotics, arabinose and caffeine molecules (100μM) at the ratio of 1:100. Use the same amount of caffeine-sensing bacteria and M9 medium plus antibiotics for serial 2-fold dilution.

2. 8 h of incubation at 25 °C

3. Fluorecence tests using plate reader with the range of wavelength 535/595.

4. Bacteria liquid concentration tests using plate reader with the wavelength of 600.

5. Plot the fluorescence testing curve.

**[in beads]**

1. Encapsulate bacteria containing caffeine sensing genetic circuit into hydrogel beads.

2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h.. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).

3. Beads containing engineered bacteria are incubated in LB plus antibiotics, arabinose (10 mM), and caffeine molecules (100 µM) at 25 °C for 12 h.

4. Test results can be analysed by fluorescence microplate assays or fluorescence microscope.

a. Fluorescence microplate assays:

i. Remove the shell of hydrogel beads using a razor blade and a tweezer.

ii. Place the hydrogel beads in a vitreous bead beater, add 0.5 mL of filtered, sterilized PBS. Homogenize hydrogel beads using bead beater.

iii. After centrifugation at 300g for 5min, 150 µL of supernatant was taken into 96-well plates, and microplate reader is used to test fluorescence in the wavelength range of 535/595 nm.

b. Fluorescence microscope: Slice the bead with a sharp razor blade at thickness of roughly 0.5 mm. The sliced sample was then imaged with a fluorescence microscope with excitation wavelength at 535 nm and emission wavelength at 595 nm.

### Stress resistance

#### Heat resistance

**[in beads]**

1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.

**2. Group A - suitable condition**

a. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 2 h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**3. Group B - thermal stimulus**

a. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 90 ℃ for 2 h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

d. Biological replication twice.

**[bacteria]**

1. Group A - suitable condition

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 2 h.

b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**2. Group B - thermal stimulus**

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 90℃ for 2 h.

b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

c. Biological replication twice.

#### Acid resistance

**[in beads]**

1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.

**2. Group A - suitable condition**

a. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 1 h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**3. Group B - acid stimulus**

a. Place two hydrogel beads in two 1.5 mL electropolished tubes. Add 500 μL LB media plus antibiotics with pH values of 1.5 and 2.5 to each tube, and incubate at 37 ℃ for 1h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

d. Biological replication twice for each pH condition.

**[bacteria]**

1. Group A - suitable condition

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 1 h.

b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**2. Group B - acid stimulus**

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media plus antibiotics with pH values of 1.5 and 2.5, incubate at 37℃ for 1 h.

b. Use 0.5 ml filtered, sterilized PBS to dissolve dry bacteria. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

c. Biological replication twice.

#### Alkali resistance

**[in beads]**

1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.

**2. Group A - suitable condition**

a. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 1 h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**3. Group B - alkali stimulus**

a. Place two hydrogel beads in two 1.5 mL electropolished tubes. Add 500 μL LB media plus antibiotics with pH values of 10 and 11 to each tube, and incubate at 37 ℃ for 1h.

b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.

c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

d. Biological replication twice for each pH condition.

**[bacteria]**

1. Group A - suitable condition

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 1 h.

b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

**2. Group B - alkali stimulus**

a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media plus antibiotics with pH values of 10 and 11, incubate at 37℃ for 1 h.

b. Use 0.5 ml filtered, sterilized PBS to dissolve dry bacteria. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.

c. Biological replication twice.

### L-dopa expression

1. Pick a single colony from streaked microbes on the plate, and cultivate at 37°C overnight.

2. Encapsulate bacteria containing the caffeine circuit into hydrogel beads, as well as beads enclosing unmodified strains for control.

3. Incubate the beads in LB plus antibiotics and caffeine molecules (100 mM) at 37 °C for 24h. The liquid medium should also contain 4.5mM of tyrosine and 1mM of IPTG.

4. Transfer 180μl of the liquid medium from each well into a 96-well plate individually, then add 20 µl of 100 mM NaIO3, leave for half an hour under room temperature to react.

5. Compare with L-dopa colour standard.

### Toxin protein expression

1. Pick a single colony with arabinose-induced toxin expression gene circuit from the plate and culture it overnight.

2. Transfer the 1:100 bacterial solution cultured overnight to 5ml of 10mM arabinose medium containing the corresponding antibiotic, and culture it overnight at 37°C in the corresponding antibiotic-free arabinose medium.

3. Dilute the overnight cultured bacteria in a 10-fold gradient in a 96-well plate, record the concentration of the undiluted bacteria as 1, and take 8 microliters each of the negative fifth power of the concentration of 1-10. Gradient dot plate counting.

4. Calculate killing efficiency.

## Competent Preparation

![](https://static.igem.org/mediawiki/2021/7/7a/T--NDNF_China--iconfv4.png)

1. Streak and culture top 10 strain with pCas plasmid overnight on kana resistant LB plate (the seeds are from competent cells).

2. Pick a single colony and culture it overnight at 30°C in 4ml LB medium.

3. The flask is cultured with shaking at 30°C until the OD600 reaches 0.4-0.5. The time required for this process depends on the type of strain. Add arabinose with a concentration of 10mM one hour before collecting the bacteria.

4. Usually after 2.5h-4h, three flasks are quickly placed on ice, shaken vigorously, and the temperature is quickly cooled.

5. For the subsequent processes: Try to carry out at low temperature (add 40g NaCl in the ice box).

6. Transfer the medium to a pre-cooled centrifuge tube at 4°C 4500rpm and centrifuge for 10 minutes, fully discard the supernatant and add 2/3 volume of pre-cooled CaCl2-MgCl2 to each tube.

7. Mix the solution, resuspend the cells, ice bath for 10 minutes at 4°C, centrifuge at 4500 rpm for 10 minutes, fully discard the supernatant.

8. Add 1/25 volume of pre-cooled 0.1M CaCl2 solution to each tube, resuspend the cells, ice bath for 10 minutes, add 7% to each tube Pre-cooled DMSO, ice bath for 10 minutes.

9. Distribute into 1.5ml EP tubes, each EP tube containing 100ul resuspension solution, and quickly freeze the cells with liquid nitrogen, then put them in a -80°C refrigerator.

## Barcode addition

![](https://static.igem.org/mediawiki/2021/a/a9/T--NDNF_China--iconfv5.png)

1. Transform pCas plasmid into top10 strain, kana resistant plate. 30℃ culture screening

2. Pick out positive clones and prepare competent clones according to the method in the previous section.

3. The chemical transformation method transforms the plasmid pTargetT containing homologous fragments and barcode into competent, and after 1 hour of recovery at 30°C, screen with kana+amp plate.

4. Verify the clones after overnight culture to verify the success of gene editing.

5. Pick a single colony that has been successfully edited in LB liquid medium (kana resistance), add 0.5mM IPTG and incubate for 8-20 hours, streak out the single bacteria, and pick single colonies and inoculate them respectively for kana resistance And kana+amp resistant medium, verify the elimination of pTargetT plasmid.

6. After verifying that the pTargetT plasmid is successfully eliminated, transfer the bacterial solution to non-resistant LB liquid medium and cultivate at 40°C to eliminate pCas.

7. Streak out the single bacteria and pick a single colony, respectively inoculate them in non-resistant and kana resistant medium to verify the elimination of pCas plasmid.

## RPA

![](https://static.igem.org/mediawiki/2021/a/a7/T--NDNF_China--iconfv6.png)

Use *colloidal gold test strip DNA rapid amplification Kit at room temperature*

Put the solution below into an environment with 39℃ for 8mins.

![](https://static.igem.org/mediawiki/2021/4/4a/T--NDNF_China--RPA_material.png)
![](https://static.igem.org/mediawiki/2021/6/68/T--NDNF_China--barcode_testing_material.png)

## Barcode testing

![](https://static.igem.org/mediawiki/2021/0/0b/T--NDNF_China--iconfv1.png)

1. Assemble the reaction at room temperature in the order above (table on the upper right):

2. Pre-incubate for 10 minutes at 25⁰C.

3. Add 3 µl of 30 nM substrate DNA (30 µl final volume).

4. Mix thoroughly and pulse-spin in a microfuge.

5. Incubate at 37°C for 10 minutes.

6. Incubate at room temperature for 10 minutes.

7. Proceed with analysis.

![](https://static.igem.org/mediawiki/2021/d/d8/T--NDNF_China--footerff.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NDNF China/Results

![](https://static.igem.org/mediawiki/2021/a/a2/T--NDNF_China--Loading.gif)

# 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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)