iGEM 2023 — Team oxford

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{% block title %}Wet Lab{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## Wet Lab

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## Design

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### The Idea:

We are aiming to use modural LucCage – LucKey system designed by Baker Lab. This new class protein biosensors use de novo design principles. These biosensors function as modular molecular devices that switch from a non-luminescent state to a luminescent state upon the binding of a target molecule. The design is simplified by requiring only one target binding domain, which allows for direct readouts in solution. The sensors are highly sensitive and can detect various clinically relevant proteins and antibodies. We decided to bring the power of this biosensor to help humanity.

### Our Binders:

Bacteriocins are strong, naturally-occuring bacterial toxins. Their N-terminal domain functions as a binder, while the C-terminal domain is highly toxic. Bacteriocins are species specific – we decided to use that property to selectively detect bacteria. Two bacteriocins were picked for testing based on their shape, binding properties and the bacterial species they target (see human practices tab)

### Targets:

**1. Escherichia coli** is a highly adaptable bacteria, E. coli strains can cause a multitude of diseases including urinary tract infections, septicemia, and neonatal meningitis. Colicin E3 - the targets would be BtuB in E. coli, outer membrane cobalamin transporter
Affinity:  Kd = 0.9 ± 0.2 nM (ka = 4.5 ± 0.1 × 105 s−1 M−1; kd = 4.1 ± 0.5 × 10−4 s−1) at pH 8, 0.1 M ionic strength. This corresponds to a free energy of binding of R135 of approximately −12 kcal mol−1

Important notes:
Because of the predominantly β-strand structure of BtuB, it was possible using far-UV circular dichroism (CD) to measure the changes in helical content of R135, caused by binding to BtuB. Upon complex formation, the helical content of R135 decreased by 12 ± 5%.

**2. Pseudomonas aeruginosa** This bacterium primarily causes respiratory and urinary tract infections, particularly in immunocompromised individuals. It is also a common cause of sepsis and burn wound infections. Key Paper:
Kurisu, Genji, et al. “The Structure of BtuB with Bound Colicin E3 R-Domain Implies a Translocon.” Nature Structural & Molecular Biology, vol. 10, no. 11, Oct. 2003, pp. 948–54, https://doi.org/10.1038/nsb997.  Pyocin S2 binding the iron transporter FpvAI. Affinity: Kd = 240 pM  Deletion of the first 45 residues, including the PRR (Δ1–45 pyoS2NTD), decreased pyoS2NTD binding ∼1,000-fold.
.

Important notes:
There is some non-specific binding to removing the ß-hairpin. Direct binding of pyoS2NTD to TonB1 was shown by ITC (Kd ∼ 1 μM), as well as by cross-linking, and was abolished when the β-hairpin was deleted (Δ1–30 pyoS2NTD)
FpvAI was expressed in E. coli TNE012 cells (ompA−, ompB−, and tsx−) transformed with pNGH183 carrying the fpvAI gene from P. aeruginosa PAO1 with an E. coli ompF signal sequence. After isolation of the OM fraction, FpvAI was purified by anion exchange chromatography and SEC.
Direct binding of pyoS2NTD to TonB1 was shown by ITC (Kd ∼ 1 μM), as well as by cross-linking, and was abolished when the β-hairpin was deleted (Δ1–29 pyoS2NTD)

### To truly help where the help is needed, our biosensor must meet some key conditions:

**Specificity:** The biosensor should accurately identify the specific bacterial strain it is designed to detect without cross-reactivity with other bacterial species or host biomolecules. This may require the careful selection and design of proteins that undergo unique conformational changes in response to target bacterial proteins or other biomarkers.

**Sensitivity:** The biosensor should be able to detect bacterial presence even at low concentrations to diagnose infections in their early stages.

**Stability:** Given the harsh conditions and lack of refrigeration in low-resource settings, the biosensor should be stable at a wide range of temperatures and humidity levels. It should also be resistant to degradation from microbial contamination or other environmental factors.

**Ease of Use:** The biosensor should be designed to be user-friendly, requiring minimal training for operation. Consider simple color change signals or other clear indicators of bacterial presence that can be easily interpreted.

**Affordability:** To be accessible in low-resource settings, the biosensor should be designed with low-cost materials and fabrication processes. (needs concrete value, e.g. < 1 USD / test)

**Scalability:** The design should be scalable for mass production and distribution without a significant increase in unit cost.

**Rapid Response:** The time from sample introduction to result should be minimized to facilitate fast diagnosis and treatment.

**Portability:** The biosensor should be compact and lightweight for easy transportation and use in field settings.

**Safety:** The biosensor should be safe to use and dispose of, with minimal risk of biohazard or chemical hazard.

**Robustness:** The biosensor should provide reliable results across a wide range of operating conditions and should not be overly sensitive to slight variations in handling or environmental conditions.

## Engineering

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Our engineering followed the design-test-build cycle. Our gene sequences came from IDT gene blocks. We cloned them on various plasmids to find the most optimal vector. We used in-Fusion cloning method – that way we could clone our genes onto linearized plasmids. As part of the design, we included overhangs to recombine with enzyme restriction sites of the

We took multiple trials to achieve successful cloning. After the attempts we were assessing success rate of cloning by antibiotic selection resistance (ampicillin or kanamycin) and performed a PCR insert check on chosen colonies before sample cultivation. We utilized pET21, as suggested by our supervisor, then, upon little success rate switched to a plasmid kindly provided to us by Baker Lab to finally settle on using pRSET A – a more unified vector. After the design we were optimizing the growth conditions for the bacterial culture, varying incubation times and IPTG concentrations to obtain best results in protein amounts. The details of our expression experiments can be found in our lab protocols. The aim was first to determinate suitable growth conditions by running a small scale expression test to then move onto a larger culture in order to grow enough protein to harvest. We engineered a His-tag as part of our proteins and could therefore use IMAC purification.

Finally, we were able to measure the luminescence of the sample. We used crude lysate samples as well as the purified protein. We mixed them with living bacteria to see if they would be detectable.
Our results were inconclusive, and more data on the project is needed. The purification steps failed, and us such we were unable to obtain reliable data. We strongly believe in our project and, with further studies, should be able to bring water safety to where it is most needed.

![](https://static.igem.wiki/teams/4932/wiki/baker-lab-cage-plasmid-map.png)
![](https://static.igem.wiki/teams/4932/wiki/baker-lab-key-plasmid-map.png)

## Wet Lab

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### Protocols

See our protocols below!

![](https://static.igem.wiki/teams/4932/wiki/apola-and-zofia-in-the-lab-13-july.jpeg)
![](https://static.igem.wiki/teams/4932/wiki/lab-photos/lab-image-1.jpeg)
![](https://static.igem.wiki/teams/4932/wiki/apola-delphine-zofia-gel-electrophoresis-17-july.jpeg)

## Results and Luminescence

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### Results

Our results were inconclusive, and more data on the project is needed. The purification steps failed, and us such we were unable to obtain reliable data. We strongly believe in our project and, with further studies, should be able to bring water safety to where it is most needed.

### Future Outlook

Our biosensor is modular, meaning it can be adapted easily – weather to serve water emergency in case of an epidemic or be adapted to a completely different purpose. As we managed to develop protocols for handling and expressing the biosensor we’re hoping that we and any other groups working on it in the future will be able to make water safety trully accessible.

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* **Specificity:** The biosensor should accurately identify the specific bacterial strain it is designed to detect without cross-reactivity with other bacterial species or host biomolecules. This may require the careful selection and design of proteins that undergo unique conformational changes in response to target bacterial proteins or other biomarkers.
* **Sensitivity:** The biosensor should be able to detect bacterial presence even at low concentrations to diagnose infections in their early stages.
* **Stability:** Given the harsh conditions and lack of refrigeration in low-resource settings, the biosensor should be stable at a wide range of temperatures and humidity levels. It should also be resistant to degradation from microbial contamination or other environmental factors.
* **Ease of Use:** The biosensor should be designed to be user-friendly, requiring minimal training for operation. Consider simple color change signals or other clear indicators of bacterial presence that can be easily interpreted.
* **Affordability:** To be accessible in low-resource settings, the biosensor should be designed with low-cost materials and fabrication processes. (needs concrete value, e.g. < 1 USD / test)
* **Scalability:** The design should be scalable for mass production and distribution without a significant increase in unit cost.
* **Rapid Response:** The time from sample introduction to result should be minimized to facilitate fast diagnosis and treatment.
* **Portability:** The biosensor should be compact and lightweight for easy transportation and use in field settings.
* **Safety:** The biosensor should be safe to use and dispose of, with minimal risk of biohazard or chemical hazard.
* **Robustness:** The biosensor should provide reliable results across a wide range of operating conditions and should not be overly sensitive to slight variations in handling or environmental conditions.

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## RESULTS

### OVERVIEW

The results outline the experimental work carried out over 1-2 months of lab work. First, we describe the construction and characterization of the various sensing modules cloned in the E-coli strain using
Snapgene. To separate the DNA fragments according to size and charge after a restriction digest, the team
simulated agarose gel electrophoresis in SnapGene. The second part presents the preliminary experiments
showing the digestion and ligation of our parts. With lots of research and iterations, the team designed
the genetic parts for our project in silico with the SnapGene application and sent them out for
synthetization. Our less experienced team with biological lab work (70% of the team are computer
science, mechanical and electrical engineering majors) started practicing in the lab with digestions,
ligations, transformations, gel electrophoresis, and making competent cells. We also explain the troubleshooting of these initial experiments
determining the issues with the ligation and digestion. Then, we explain the corrections of the digestion and ligation protocols resulting from this troubleshooting.
Also, we present the results obtained from the final try, continued with miniprep, PCR and gel electrophoresis to characterize the part. And finally, the result for the
first try of the hydrogel encapsulation and some proposed future experiments to obtain the expected results.

### PRELIMINARY SIMULATIONS

Before performing this experiment in the Lab, the team utilized Snapgene to simulate the digestion and ligation of the various parts. In this approach, we first performed a linear ligation between the sensing module and kill switch fragments (Au and UV, As and UV, Fe part 1 & 2 and UV) to evaluate if it's feasible to be ligated. This was done successfully, as represented in figures (1a,2a, and 3a). We used restriction and insertion cloning to insert these fragments into the plasmid (pUC 19). By inserting, we cut the plasmid with the EcoR1 and Pst1 to create sticky ends to facilitate ligation. We also cut the fragments with EcoR1 and PSt1 to maximize ligation efficiency. This resulted in complete plasmids with various sensing module inserts, as shown in figures 1b, 2b, and 3b. Figure 1c. 2c and 3c depict the vectors and inserted fragments with their various sizes (base pairs), which enabled the team to visualize how the insertion was done and the size of the various components.

1. Au and Kill Switch Model

![](https://static.igem.wiki/teams/4186/wiki/fig-1a.jpg)

Figure 1a: Linear ligation of gold inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-1b.jpg)

Figure 1b: Ligated plasmid with Au-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-1c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-1c2.jpg)

Figure 1c: Illustration depicting the vector (pUC 19) and the fragment (Au inducer-UV)

 2. As and Kill switch Module

![](https://static.igem.wiki/teams/4186/wiki/fig-2a.jpg)

Figure 2a: Linear ligation of arsenic inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-2b.jpg)

Figure 2b: Ligated plasmid with As-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-2c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-2c2.jpg)

Figure 2c: Illustration depicting the vector (pUC 19) and the fragment (As inducer-UV)

 3. Fe and Kill switch Module

![](https://static.igem.wiki/teams/4186/wiki/fig-3a.jpg)

Figure 3a: Linear ligation of ferric inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-3b.jpg)

Figure 3b: Ligated plasmid with As-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-3c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-3c2.jpg)

Figure 3c: Illustration depicting the vector (pUC 19) and the fragment (Ferric inducer-UV)

### GEL ELECTROPORESIS

In this simulation, the team wanted to identify the inserted fragments that bear the inducers or sensing modules. From the ligation and cloning, the expected sizes for Fe, Au and As-UV fragments were 2455bp, 2368bp and 2324bp, respectively. These sizes were checked with the 1% agarose gel electrophoresis, where the NEB 1 kb DNA ladder was used for the simulation to quantify the inserts and the DNAs. From figure 4, using the ladder marker at 3.0kb, we obtained the expected size as shown. From here, we concluded that since the simulation works, we could go ahead and do the lab experiments to confirm what is expected from the simulations.

![](https://static.igem.wiki/teams/4186/wiki/fig-4.jpg)

Figure 4: Identifying fragments that bear the inserted genes (Au, Fe and As inducers) by gel electrophoresis simulation.

### WET LAB EXPERIMENTS AND RESULTS

Once the parts arrived, we went ahead with digestion of our genetic parts or genes of interest (Gold [Au], Arsenic [As] and Iron [Fe (I & II)] sensing modules, and the autolytic UV kill switch modules) and the vector we chose for our design (Puc19 Plasmid). We continued with ligating the different gene fragments with the vector and performed transformation with the chassis we chose (Escherichia coli). While doing these experiments, we also made competent cells from bacteria culture that were grown overnight.

![](https://static.igem.wiki/teams/4186/wiki/fig-5.png)

Figure 5: Infographics showing the experimental processes that were undertaken

1.1 DIGESTION

We made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part.
After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

![](https://static.igem.wiki/teams/4186/wiki/digestion.svg)

After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

1.2 LIGATION

We made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part.
After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

![](https://static.igem.wiki/teams/4186/wiki/ligation.svg)

After ligation, we transformed the parts from the three tubes from ligation with the competent cells and allowed for overnight growth.

1.3 OBSERVATION, RESULTS AND TROUBLESHOOT

After the overnight growth, we did not observe any growth or colonies on our agar plates for Arsenic and Fe sensing modules. However, we observed very few colonies for the Au sensing module. We took the colony and added it to ampicillin media, added some gold particle-chippings and grew it overnight. We plated the culture with the gold particles on ampicillin plates to observe growth and color change over a period. No color change was observed for this investigation, however.
We then suspected something might have been wrong with the concentration of the plasmid used. We suspected the plasmid might have been over diluted to undergo digestion and ligation. Thus, we sought to find out if our suspicion was correct. We performed a digestion, ligation, and transformation for only the puc19 plasmid to obtain a very concentrated plasmid after we conducted a mini prep. After the mini prep, we ran a gel, to confirm if we were correct. From the gel image shown below we were correct. The second well which had the initial diluted plasmid DNA had no trace of DNA on its column, while the next four wells which had the concentrated plasmid had traces of DNA on their columns.

![](https://static.igem.wiki/teams/4186/wiki/fig-6.jpg)

Figure 6: Gel electrophoresis results comparing two different plasmids obtained with different miniprep kits to the NEB 1kb MW ladder

Another suspicion we had about the results we had was whether the mini-prep kit we used was problematic. Thus, for the gel image shown above, we conducted two mini-prep with an old and a new kit. The columns labelled A1 and A2 were minipreps conducted with the new kit and the columns labelled B1 and B2 were minipreps conducted with the old kit. We had two columns for each kit used because we used two different colonies for each kit. Based on the results from the gel, we concluded that both our old and new mini-prep kits had no issues.

1.4 PLASMID CONCENTRATION TROUBLE SHOOT

We were able to troubleshoot what was wrong and realized the plasmid DNA we used initially was very diluted. After obtaining the concentrated plasmid DNA, we went ahead to ascertain what concentration of the current plasmid DNA used would be ideal for our experiments. We diluted the current concentrated plasmid DNA into three concentrations (1:2 concentration, 1:5 concentration and a 1:10 concentration). We ran a gel and based on the results from the gel image below, we chose to go with a 1:10 concentration for our subsequent experiments.

![](https://static.igem.wiki/teams/4186/wiki/fig-7.jpg)

Figure 7: Gel electrophoresis results comparing different pUC 19 ratios

1.5 DIGESTION AND LIGATION TROUBLE SHOOT

The initial results obtained also raised concerns about whether we had issues with the digestion and ligation of our parts. Thus, before we went ahead with the second lab experiment with the remaining of our synthesized parts, we wanted to ascertain if our digestion and ligation worked with no errors. As a result, we performed digestion and ligation with the 1:10 concentrated plasmid DNA from the previous experiment. We digested with the restriction enzymes, SpeI and XbaI since both when used to cut the vector would have sticky ends that are compatible. For this experiment, we used two different approaches to digestion. One digestion approach involved making a Mastermix made up of the reagents used in digestion and the other approach involved no Mastermix. We suspected that making a Mastermix before digestion might further dilute the concentrations of the restriction enzymes and the DNA parts. Thus, we conducted these experiments to make sure our suspicions were correct or otherwise.

For this experiment, we had four digestions of pUC 19. Two of these digestions were done using the Mastermix approach (MM) and the other two were digested using the No Mastermix approach (No MM). One of the digested Puc19 part tubes for each digestion approach were transformed directly while the other two for each digestion approach went ahead to ligation.

We figured out that, if digestion worked then, the transformation for the digested parts from each approach should have no growth when plated on the ampicillin agar plates. The reasoning behind this is that, if the plasmids were not correctly cut or digested, the bacteria would have full plasmids with ampicillin resistance which would mean that these bacteria would not be killed by the ampicillin antibiotic. However, if they were cut correctly, the bacteria would not have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plate and thus would experience no growth.

We further ligated the other two tubes, one each from the two digestion approaches, transformed with competent cells, and allowed for growth overnight. We had two control experimental setups for this experiment:

➕ Positive control (transformed E. coli bacteria with undigested and unligated Puc19 plasmid)

➖ Negative control (transformed E. coli bacteria only)

The positive control is expected to have growth because the E. coli bacteria would have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plates. The negative control however is expected not to have any growth since it has no plasmid with ampicillin resistance and will be destroyed by the ampicillin antibiotic from the agar plate.

![](https://static.igem.wiki/teams/4186/wiki/fig-8.png)

Figure 8: results obtained from Digestion and ligation troubleshoot (a) transformed digested plasmid with master mix (b) transformed digested plasmid without master mix (straight NEB protocol) (c) Transformed ligated plasmid with master mix (d)Transformed ligated plasmid with no master mix (e) positive control (pUC 19 + competent cells) (f) Negative control (Just competent cells with no plasmid or DNA)

From the results we obtained after transformation as seen above, we had growth for the following experimental setups:

- Transformation using direct digested Puc19 from the Mastermix D(MM) digestion approach

- Transformation using digested and ligated Puc19 from the Mastermix digestion approach L(MM)

- Transformation using digested and ligated Puc19 from the No Mastermix digestion approach L(No MM)

- Positive control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid)

From the results we obtained after transformation as seen above, we had no growth for the following experimental setups:

- Transformation using direct digested Puc19 from the No Mastermix (No MM) digestion approach

- Negative control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid

1.6 CONCLUSION

This lab experiment we performed gave us the results that we envisioned. The results implied that the Mastermix digestion approach that we used did not ensure that all or some of the plasmids were digested, hence the growth from the direct transformation. We then concluded that using the No Mastermix approach for digestion for future lab experiments would be ideal.

2.0 SECOND LAB EXPERIMENT WITH SYNTHESIZED PROJECT PARTS

We conducted the second lab experiment with the rest of the synthesized parts for our project. We used the 1:10 concentrated plasmid, No Mastermix approach for digestion and made new competent cells for this last experiment with the final set of the synthesized parts.

We used the same reagents and micro volumes from the first lab experiment conducted with synthesized genetic parts for the project.

The results we obtained from the transformations are displayed below.

![](https://static.igem.wiki/teams/4186/wiki/fig-9.png)

Figure 9: Results obtained from Digestion and ligation of synthesized IDT and Twist parts (a) Gold inducer – Kill switch module (b) Arsenic inducer – Kill switch module (c) Ferric Inducer module

From the results displayed above, we observed a few colonies but still with low transformation efficiency. However, we took some of these colonies and grew them in the media overnight. We then used this overnight culture to perform a mini prep. After the mini-prep, we performed a Polymerase chain Reaction to amplify the inserted genes for each of the synthesized DNA in the Puc19 plasmid.

2.1 PCR EXPERIMENT

For the PCR, we had two tubes from two colonies of the Fe (I & II), two tubes from two colonies of Au and three tubes from three colonies of the As. We included a Puc19 plasmid DNA as control. Below are tables for PCR conditions and PCR reagent volumes we used.

![](https://static.igem.wiki/teams/4186/wiki/pcr-conditions.svg)

![](https://static.igem.wiki/teams/4186/wiki/pcr-reagents.svg)

2.2 GEL ELECTROPHORESIS

After the PCR, we run a gel with the amplified DNA parts. Below is a gel image of the gel run on the PCR amplified genetic parts.

![](https://static.igem.wiki/teams/4186/wiki/fig-10.jpg)

Figure 10: Gel electrophoresis results obtained for amplified DNA parts

2.3 CONCLUSION AND TROUBLESHOOTING

The anticipated band size for the genetic inserts used in this PCR gel are listed below.

![](https://static.igem.wiki/teams/4186/wiki/band-sizes.svg)

From the gel image above, it can be clearly observed that none of our genetic inserts were present (columns 2-8). However, the amplified portion of the puc19 in the PCR, which had approximately 184 base pairs, was present in each of the genetic inserts.
We suspected that one of the reasons the result came out the way it did might be because of the annealing temperature used for the PCR which was very close to the TM of the primers, impeding the amplification of the insert. Other reasons could be that the ligation of the parts might not have worked properly. We think that this is a possibility because the number of colonies was small. Since the plasmid cannot ligate itself we think that maybe few plasmids were not digested and lead to small transfection efficiency.

To trouble shoot if the PCR did not work correctly, we aimed to run another gel with the DNA parts directly from the mini prep. Unfortunately, we ran out of SYBR gold stain, used to visualize the DNA in the gel. We ordered some SYBR gold, but it did not arrive before the deadline to perform the experiment and hence could not troubleshoot this reasoning.

### FUTURE WORKS

📝 Once we have DNA staining dye, we would change the annealing temperature to increase the difference between the annealing temperature and TM .

📝 We would order new genetic parts for our project and troubleshoot digestion and ligation of the parts with the Puc19 plasmid.

### HYDROGEL ENCAPSULATION

Our solution proposed the deployment of live E. coli cells in the environment, so we ensured biosecurity and safety while maximizing the efficiency of our biosensor through hydrogel encapsulation. During the project's progress, the team moved from theoretical research to experimental lab work to test the theory of hydrogel1 encapsulation. Below is the protocol used and the results obtained. We could not add the hard shell of polyacrylamide hydrogel because our order did not arrive in time. Alginate forms hydrogel in di-cationic solutions (e.g., Ca2+, Ba2+) and has been used for various biomedical applications.

![](https://static.igem.wiki/teams/4186/wiki/fig-11.png)

Figure 11: Cell encapsulation in hydrogel capsules (a) schematic of the DEPCOS platform. (b) The process of core shell encapsulation of cells. Droplets of 2.5% alginate with engineered E. coli were crosslinked in a CaCl2 solution to form the soft core of the bead. Outsourced from (Tang, Tham, Liu, & Lu, 2020)

Manufacturing the Alginate Cores

A 10% alginate solution was made by dissolving medium viscous alginate in Distilled water, followed by autoclaving at 120 degrees to ensure sterility. A fresh bacterial culture with GFP and Red Mcherry was then mixed with the alginate solution in a one-to-one volume ratio to reach a final alginate concentration of 5 w.t%. This bacteria alginate premix was loaded into a syringe and dropwise added into a beaker of a CaCl2 solution (Fig. 11a) to form a beadlike droplet (Fig. 11b) with controllable diameters ranging from 2.5mm to 3mm. Immersing the droplet in the CaCl2 solution for a few minutes solidified the droplets formed.

![](https://static.igem.wiki/teams/4186/wiki/fig-12.png)

Figure 12: (left) The process of core shell encapsulation of cells. 2.5% alginate solution (right) Droplets of engineered E. coli crosslinked in a CaCl2 solution to form the soft core of the hydrogel

![](https://static.igem.wiki/teams/4186/wiki/fig-13.png)

Figure 13: Hydrogel containing E coli cells with (a) alginate containing cells with GFP (b) alginate containing cells with Red mcherry (c) Alginate

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