iGEM 2021 — Team IISER_Kolkata

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# Team:IISER Kolkata/Notebook

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1. [Home](https://2021.igem.org/Team%3AIISER_Kolkata)
2. Team

   * [Team Members](https://2021.igem.org/Team%3AIISER_Kolkata/Team)
   * [Attributions](https://2021.igem.org/Team%3AIISER_Kolkata/Attributions)
   * [Collaborations](https://2021.igem.org/Team%3AIISER_Kolkata/Collaborations)
   * [Partnership](https://2021.igem.org/Team%3AIISER_Kolkata/Partnership)
3. Project

   * [Description](https://2021.igem.org/Team%3AIISER_Kolkata/Description)
   * [Design](https://2021.igem.org/Team%3AIISER_Kolkata/Design)
   * [Contribution](https://2021.igem.org/Team%3AIISER_Kolkata/Contribution)
   * [Engineering](https://2021.igem.org/Team%3AIISER_Kolkata/Engineering)
   * [Improvement](https://2021.igem.org/Team%3AIISER_Kolkata/Improve)
4. Wet Lab

   * [Experiments](https://2021.igem.org/Team%3AIISER_Kolkata/Experiments)
   * [Notebook](https://2021.igem.org/Team%3AIISER_Kolkata/Notebook)
   * [Parts](https://2021.igem.org/Team%3AIISER_Kolkata/Parts)
   * [Safety](https://2021.igem.org/Team%3AIISER_Kolkata/Safety)
   * [Results](https://2021.igem.org/Team%3AIISER_Kolkata/Results)
5. Dry Lab

   * [Mathematical Modeling](https://2021.igem.org/Team%3AIISER_Kolkata/Model)
   * [Molecular Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/Molecular_Dynamics)
   * [Gene Regulation](https://2021.igem.org/Team%3AIISER_Kolkata/GeneRegulation)
   * [Biofilm Degradation](https://2021.igem.org/Team%3AIISER_Kolkata/Biofilm_Degradation)
   * [Population Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/PopulationDynamics)
6. Human Practices

   * [Integrated Human Practices](https://2021.igem.org/Team%3AIISER_Kolkata/Human_Practices)
   * [Proposed Implementation](https://2021.igem.org/Team%3AIISER_Kolkata/Implementation)
   * [Communication](https://2021.igem.org/Team%3AIISER_Kolkata/Communication)
7. Awards

   * [Education](https://2021.igem.org/Team%3AIISER_Kolkata/Education)
   * [Inclusivity](https://2021.igem.org/Team%3AIISER_Kolkata/Inclusivity)
   * [Supporting Entrepreneurship](https://2021.igem.org/Team%3AIISER_Kolkata/Entrepreneurship)
8. [Medals](https://2021.igem.org/Team%3AIISER_Kolkata/Medals)

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* Overview
* Wet Lab Notebook
* Human Practices Notebook
* Dry Lab Notebook

**overview**

All the notebooks were maintained in Benchling throughout the project.

**Wet Lab Notebook**

If you can not view above PDF: [DOWNLOAD PDF](https://static.igem.org/mediawiki/2021/1/13/T--IISER_Kolkata--pdf-Notebook-WetLabWorkflowBenchling.pdf)

**Human Practices Notebook**

If you can not view above PDF: [DOWNLOAD PDF](https://static.igem.org/mediawiki/2021/5/57/T--IISER_Kolkata--Human_Practices_Workflow_%C2%B7_Benchling_%281%29.pdf)

**Dry Lab Notebook**

If you can not view above PDF: [DOWNLOAD PDF](https://static.igem.org/mediawiki/2021/4/47/T--IISER_Kolkata--pdf-Notebook-DryLabWorkflowBenchling.pdf)

**Contact us at**

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# Team:IISER Kolkata/Engineering

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1. [Home](https://2021.igem.org/Team%3AIISER_Kolkata)
2. Team

   * [Team Members](https://2021.igem.org/Team%3AIISER_Kolkata/Team)
   * [Attributions](https://2021.igem.org/Team%3AIISER_Kolkata/Attributions)
   * [Collaborations](https://2021.igem.org/Team%3AIISER_Kolkata/Collaborations)
   * [Partnership](https://2021.igem.org/Team%3AIISER_Kolkata/Partnership)
3. Project

   * [Description](https://2021.igem.org/Team%3AIISER_Kolkata/Description)
   * [Design](https://2021.igem.org/Team%3AIISER_Kolkata/Design)
   * [Contribution](https://2021.igem.org/Team%3AIISER_Kolkata/Contribution)
   * [Engineering](https://2021.igem.org/Team%3AIISER_Kolkata/Engineering)
   * [Improvement](https://2021.igem.org/Team%3AIISER_Kolkata/Improve)
4. Wet Lab

   * [Experiments](https://2021.igem.org/Team%3AIISER_Kolkata/Experiments)
   * [Notebook](https://2021.igem.org/Team%3AIISER_Kolkata/Notebook)
   * [Parts](https://2021.igem.org/Team%3AIISER_Kolkata/Parts)
   * [Safety](https://2021.igem.org/Team%3AIISER_Kolkata/Safety)
   * [Results](https://2021.igem.org/Team%3AIISER_Kolkata/Results)
5. Dry Lab

   * [Mathematical Modeling](https://2021.igem.org/Team%3AIISER_Kolkata/Model)
   * [Molecular Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/Molecular_Dynamics)
   * [Gene Regulation](https://2021.igem.org/Team%3AIISER_Kolkata/GeneRegulation)
   * [Biofilm Degradation](https://2021.igem.org/Team%3AIISER_Kolkata/Biofilm_Degradation)
   * [Population Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/PopulationDynamics)
6. Human Practices

   * [Integrated Human Practices](https://2021.igem.org/Team%3AIISER_Kolkata/Human_Practices)
   * [Proposed Implementation](https://2021.igem.org/Team%3AIISER_Kolkata/Implementation)
   * [Communication](https://2021.igem.org/Team%3AIISER_Kolkata/Communication)
7. Awards

   * [Education](https://2021.igem.org/Team%3AIISER_Kolkata/Education)
   * [Inclusivity](https://2021.igem.org/Team%3AIISER_Kolkata/Inclusivity)
   * [Supporting Entrepreneurship](https://2021.igem.org/Team%3AIISER_Kolkata/Entrepreneurship)
8. [Medals](https://2021.igem.org/Team%3AIISER_Kolkata/Medals)

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* Research
* Imagine
* DNaseI
* AIP-I

**Research**

We came to know about the severe problems of Bovine mastitis from a very personal perspective through one of our teammates. After encountering the problem, detailed human practices research helped us to realize the real-life problems faced by the stakeholders and the solution they need to tackle this problem at this moment. We felt the necessity of the human-centric approach to detect and cure bovine mastitis and also be aware the people about prevention practices. From the various inputs from our stakeholders, we understood that a non-antibiotic method of treatment is the need of the hour. Antimicrobial resistance in pathogens is an alarming issue and pathogens causing bovine mastitis are also becoming antibiotic resistant due to extreme overuse of broad-spectrum antibiotics in the dairy industry. Currently, only antibiotics are used to treat
bovine mastitis disease and are not much effective for its purpose. Therefore the non-antibiotic method of treatment is needed to reduce the use of antibiotics in the dairy industry. We decided to approach this issue using synthetic biology. To ensure effective implementation of the treatment technique we also realized the need for an early, easy-to-use, cheap detection kit to detect the subclinical bovine mastitis disease.

**Imagine**

Through our interactions with dairy farmers and veterinary doctors from various parts of India, we narrowed down our research to the major reasons why Mastitis still remains a threat. The current method for detection can only detect bovine mastitis at a very later stage and is not specific. By that time it gets difficult to cure the disease. The current treatment which highly depends on broad-spectrum antibiotics is not only delivered in sufficient amounts to kill the pathogens,(S aureus and S uberis) but also could lead to antimicrobial resistance(AMR). Hence we decided to treat the disease at its root by using a combinatorial approach of early detection along with a proper treatment method that is not only the best alternative to antibiotics but also more specific and efficient.

The problem with using broad-spectrum antibiotics in the treatment of Bovine mastitis is that it leads to the development of antibiotic resistance among bacterial strains. Also most of the time, the antibiotic supplied to the udder via intramammary injection does not reach the bacteria insufficient amount. We have taken special care to address both these issues by focussing on finding the best alternative and ensuring that it gets delivered at the location of bacteria insufficient amount.

**DNaseI**

**Design**

Literature on Bovine mastitis treatment led us to discover the potential candidates for our approach. We choose to incorporate Nisin PV, a synthetic variant of Nisin A lantibiotic alongside DNaseI to eradicate S aureus and S uberis which are the major bacterias present in the udder of infected cows. Nisin PV creates pores in the bacterial membrane. DNaseI favors this activity by degrading the extracellular DNA in bacterial biofilm. DNaseI treated bacterial colony is found to be more susceptible to the activity of antibacterial agents.

Due to the time and space constraints, we decided to focus on DNaseI. Our team designed a very simple and elegant method to clone and characterize Bovine DNaseI.

**Cycle-1**

**Build and test**

Primarily, the Bovine DNaseI gene fragment was cloned and expressed in E Coli BL21 in the PSB1C3 vector. Transformants were tested using colony PCR. Expression was induced under IPTG. *See [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run1Section) for more info.*

**Learn and improve**

Unfortunately, due to COVID restrictions, we lack time and lab access for the proper extraction and purification of protein. So as an alternative, we further depended on industrially prepared DNaseI from sigma Aldrich(D2025-15KU) to carry out further experiments.

**Cycle-2**

**Build and test**

To further study the activity of DNase1 against biofilm of bacteria, we conducted a biofilm assay in 96 well plates. Before studying DNaseI activity against biofilm, we decided to standardize the biofilm formation in 96 well plates by following standard procedure. We used Pseudomonas aeruginosa for developing biofilm. Initially, bacteria were grown by varying growth media, incubation times, dilutions, and other conditions to determine the optimal conditions for maximum yield of biofilm. The yield of biofilm was calculated by determining A595 nm after crystal violet treatment.

![Engineering-001.png](https://static.igem.org/mediawiki/2021/5/57/T--IISER_Kolkata--image-Engineering-001.png)

96 Well plate showing biofilm formation after 72hr incubation, in different dilutions of different dilutions.

![Engineering-002.png](https://static.igem.org/mediawiki/2021/b/bb/T--IISER_Kolkata--image-Engineering-002.png)

Crystal Voilet added in each well after washing out excess excess cells.

![Engineering-003.png](https://static.igem.org/mediawiki/2021/2/25/T--IISER_Kolkata--image-Engineering-003.png)

96 well plate after washing off the crystal violet.

**Learn and improve**

Biofilm yield for all different conditions was determined. Based on the results the following inferences were made about conditions favouring maximum yield: see [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run1Section) for more info.

1. NB media gave higher biofilm compared to LB and TSB Media.
2. 72 hr incubation was optimal for maximum biofilm formation when compared to 24 hr and 48 hr.
3. Dilution of primary culture to 0.1 OD before adding into wells gave maximum biofilm when compared to 0.05 OD.

Hence, for further assays, we decided to incubate bacteria for 72 hrs in NB media maintaining initial OD of 0.1.

**Cycle -3**

**Build and test**

The next step is to determine the optimum concentration of DNase1 required for maximum degradation of biofilm produced. This value is critical while designing the diagnostic tool. This was determined by carrying out a biofilm assay similar to what was mentioned above. However, here we pre-incubate the media with varying concentrations of DNaseI to determine which concentration gave maximum degradation at the end of 72 hr. The degradation rate was compared in terms of biofilm percentage reduction(BPR) which is given by:

$$ BPR = \left( \frac{Control \ A595 nm - test \ A595 nm}{Control \ A595 nm} \right) \times 100 $$

**Learn and improve**

Based on the experiments, a concentration of 10 µg/ml was found to give the maximum degradation rate. This concentration was chosen as a standard in further experiments. See [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run1Section) for more info.

**Cycle-4**

**Build and test**

Considering the optimal concentration of DNaseI required for maximum degradation of biofilm, we further tried to determine the optimal time or the time for maximum degradation of biofilm. For this experiment, we developed a biofilm of Pseudomonas aeruginosa in 96 well plates for 72 hrs. Following this, all the bacteria in the well was decanted and the pure biofilm was further treated with media containing 10 µg/ml of DNaseI(concentration determined from pretreatment above) and incubated for different time intervals. Biofilm quantification was carried out and the degradation rate was compared by calculating biofilm percentage reduction(BPR).

**Learn and improve**

Maximum biofilm degradation was observed in wells incubated for 40 min.No further change was observed for a longer duration of incubation. see [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run1Section) for more info.

**AIP-I**

**Design**

To ensure that our bacteria gets activated only in the presence of our target pathogen, we decided to utilize the Quorum sensing pathway of S.aureus. S.aureus produces AIP-I as a quorum-sensing molecule. So, we choose to incorporate the agrA\_agrC system to enable our GMO to sense AIP-I. To regulate Nisin PV and DNase I production, their corresponding genes were designed to be downstream of the AIP-I inducible P2 promoter. To characterize our genetic circuits, we required AIP-I molecules. There were two options to obtain them - 1) Obtain synthetic AIP-I from a vendor, 2) Express AIP-I in bacteria. We found the second option more feasible for our team, and hence, we decided to clone the AIP-I producing Gene in E.Coli DH5α,pSB1C3 vector and use the expressed protein for our experiment.

We decided to use the parts from the iGEM DNA Distribution kit plate to assemble our gene circuit for AIP-I production. We used the IPTG inducible Plac promoter (BBa\_R0010) with the AIP generator - agrB agrD system (I746001). The Plac promoter was used as it is strongly regulated by IPTG and hence the expression of the protein can be controlled. We planned to clone the Circuit in the pSB1C3 plasmid vector itself and express them in E.Coli BL21 for protein expression.

Cycle - 1

**Build**

As we planned to use the parts from the iGEM DNA Distribution kit, it was more feasible for us to use the biobrick 2A assembly method for cloning our genetic circuit. To obtain the Plac promoter gene we did double restriction digestion with E.CoRI and Spel. The agrB agrD containing pSB1C3 plasmid was double restriction digestion using EcoRI and XbaI. The time required for complete double restriction digestion was optimized.

![Engineering-abc.png](https://static.igem.org/mediawiki/2021/2/2d/T--IISER_Kolkata--image-Engineering-abc.png)

**Test**

Optimum digestion was observed with 4 hours of incubation. The digested products were run in agarose gel electrophoresis against the DNA ladder to check the presence of digested DNA fragments. No band were observed for Plac fragments.

**Learn and Improve**

The band for Plac was not obtained in the gel as the size of the digested fragment is approximately 200bp so, it was not retained in the 0.8% agarose gel, and hence it was not visible. We learned that we should do double restriction digestion such that the desired fragments are not smaller than 800bp, to enable proper resolution of fragments in agarose gel.

**Cycle - 2**

**Build**

We modified the experimental design. This time we double digested the agrB\_agrD containing plasmids with Xbal and PstI to create the insert, while the Plac containing plasmid was digested with Spel and PstI to create a linearized plasmid. The length of both the DNA fragments was above 800bp.

![Engineering-efg.png](https://static.igem.org/mediawiki/2021/c/c5/T--IISER_Kolkata--image-Engineering-efg.png)

**Test**

The digested product was run in agarose gel to check the presence of the digested products. The desired DNA bands were obtained (see [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run2Section) for more info.). The digested fragments were purified by agarose gel extraction. The concentration of the DNA sample was checked. The concentration of the agrB\_agrD fragment was observed to be very low and the 280/260 ratio was not between 1.8 and 1.9. (Table)

|  |  |  |
| --- | --- | --- |
| Sample | Concentration (ng/ul) | 280/260 ratio |
| agrB\_agrD digested samp01 | 4.7 | 2.35 |
| agrB\_agrD digested samp02 | 3.5 | 2.06 |

**Learn and Improve.**

For restriction digestion, we used the same weight of both the plasmids samples. The plasmid containing agrB\_agrD had higher base pairs (2966bp) compared to the plasmid with Plac (2470bp). Hence we hypothesize that fewer units of agrBD plasmids were digested as compared to the Plac containing plasmids. However, For ligation, we need a higher amount of insert fragments compared to the linearized plasmid. Hence, we could not proceed with the purified digested fragments.

We understood that we needed to obtain the agrB\_agrD fragment in a much higher concentration than the Plac-containing linearized plasmid.

**Cycle - 3**

**Build**

To obtain the agrB\_agrD fragments in higher concentration, the agrB\_agrD plasmid was PCR amplified using the biobrick forward and biobrick reverse primers. The amplified fragments were then purified by PCR clean-up.

**Test**

DNA concentration of PCR amplified products was measured. A high concentration of DNA was obtained.

|  |  |  |
| --- | --- | --- |
| Sample | Concentration (ng/ul) | 280/260 ratio |
| agrB\_agrD PCR amplified samp01 | 73.7 | 1.90 |

The amplified fragments were double restriction digested. The double digested product was ligated and transformed into E.Coli DH5α. Colony PCR was performed using vf2 and VR primers. The colonies having correct (desired) plasmid were identified (see [results](https://2021.igem.org/Team%3AIISER_Kolkata/Results#run2Section) for more info). The cloned plasmid was extracted and transformed in BL21. Protein expression was induced with IPTG on reaching 0.6 OD (600 nm). The cells were pellet down, and the media was sterilized by filter sterilization. The expressed protein was concentrated by lyophilization.

The presence of the AIP molecule was checked by the induction of the GFP production under the AIP inducible P2 promoter in unidirectional circuit (BBa\_K1022100).

**Learn**

AIP molecules were successfully expressed.

### References

1. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/>
2. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867557/>
3. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288126/>
4. <https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/213/541/d5025dat.pdf>
5. <https://www.frontiersin.org/articles/10.3389/fmicb.2020.00813/full>

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# Team:IISER Kolkata/Experiments

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1. [Home](https://2021.igem.org/Team%3AIISER_Kolkata)
2. Team

   * [Team Members](https://2021.igem.org/Team%3AIISER_Kolkata/Team)
   * [Attributions](https://2021.igem.org/Team%3AIISER_Kolkata/Attributions)
   * [Collaborations](https://2021.igem.org/Team%3AIISER_Kolkata/Collaborations)
   * [Partnership](https://2021.igem.org/Team%3AIISER_Kolkata/Partnership)
3. Project

   * [Description](https://2021.igem.org/Team%3AIISER_Kolkata/Description)
   * [Design](https://2021.igem.org/Team%3AIISER_Kolkata/Design)
   * [Contribution](https://2021.igem.org/Team%3AIISER_Kolkata/Contribution)
   * [Engineering](https://2021.igem.org/Team%3AIISER_Kolkata/Engineering)
   * [Improvement](https://2021.igem.org/Team%3AIISER_Kolkata/Improve)
4. Wet Lab

   * [Experiments](https://2021.igem.org/Team%3AIISER_Kolkata/Experiments)
   * [Notebook](https://2021.igem.org/Team%3AIISER_Kolkata/Notebook)
   * [Parts](https://2021.igem.org/Team%3AIISER_Kolkata/Parts)
   * [Safety](https://2021.igem.org/Team%3AIISER_Kolkata/Safety)
   * [Results](https://2021.igem.org/Team%3AIISER_Kolkata/Results)
5. Dry Lab

   * [Mathematical Modeling](https://2021.igem.org/Team%3AIISER_Kolkata/Model)
   * [Molecular Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/Molecular_Dynamics)
   * [Gene Regulation](https://2021.igem.org/Team%3AIISER_Kolkata/GeneRegulation)
   * [Biofilm Degradation](https://2021.igem.org/Team%3AIISER_Kolkata/Biofilm_Degradation)
   * [Population Dynamics](https://2021.igem.org/Team%3AIISER_Kolkata/PopulationDynamics)
6. Human Practices

   * [Integrated Human Practices](https://2021.igem.org/Team%3AIISER_Kolkata/Human_Practices)
   * [Proposed Implementation](https://2021.igem.org/Team%3AIISER_Kolkata/Implementation)
   * [Communication](https://2021.igem.org/Team%3AIISER_Kolkata/Communication)
7. Awards

   * [Education](https://2021.igem.org/Team%3AIISER_Kolkata/Education)
   * [Inclusivity](https://2021.igem.org/Team%3AIISER_Kolkata/Inclusivity)
   * [Supporting Entrepreneurship](https://2021.igem.org/Team%3AIISER_Kolkata/Entrepreneurship)
8. [Medals](https://2021.igem.org/Team%3AIISER_Kolkata/Medals)

![Experiments-Hero.png](https://static.igem.org/mediawiki/2021/3/34/T--IISER_Kolkata--image-Experiments-Hero.png)

[![IGEM-IISERKolkata](https://static.igem.org/mediawiki/2021/c/cd/T--IISER_Kolkata--image-igemIISERK_home.png)](https://2021.igem.org/Team%3AIISER_Kolkata)
[![Namooste](https://static.igem.org/mediawiki/2021/c/c5/T--IISER_Kolkata--images-namoosteTransparent.jpg)](https://2021.igem.org/Team%3AIISER_Kolkata)

* Materials
* Competent Cells Preparation
* Cloning’s Method
* HiFi DNA Assembly
* Transformation Protocol
* Quantitative Assays

**Materials**

**Plasmids

+**

**pUC19:**
This is a high copy number plasmid. The origin of replication of this plasmid is pMB1 (High Copy Mutant). This contains Ampicillin resistance gene. This plasmid was mainly used for determining the transformation efficiency of our competent cells. We got this plasmid from one of our sponsors
[NEB](https://www.neb-online.fr/en/).

**pSB1C3:**
pSB1C3 vector is a high copy number plasmid which carries Chloramphenicol resistance gene. The origin of replication is pUC19 derived pMB1. We got this plasmid from iGEM headquarters as this plasmid is a common vector for all the Biobricks present in iGEM-21 kit plates. We used this plasmid as a vector for all the cloning and assemblies.

**List of DNA fragments ordered from Twist Bioscience

+**

**Eicsm6 Coding Region:**
GAATTCGCGGCCGCTTCTAGATGAAAATCCTGTTCTCTCCTATAGGAAATACGGACCCGTGGCGGAACGATCGAGACGGCGCAATGTTACATATTGTACGACATTATCAGCCCGATCGCGTTGTTTTGTTCTTCACTGAAAGCATATGGCAGGGCAACCAGCATTTCTCTGGGCAGCAGGCATTCGACTGGGTCAAGATTATACAATCAATCAATGAAAACTGCCAAATAGAGATCAAGTGCGACACGATAGAAGTCGAAAACGACTTTGACGCTTATAAGGACTTATTTCACCAATATCTCGTTGAAGAGAAGCGCAAGTACCCAAACGCCGAGATCTTTCTGAACGTAACCTCAGGTACCCCACAAATGGAGACGACACTGTGTCTCGAGTACGTAACCTATCCGGATAAGATGAGATGTATCCAAGTGAGTACTCCACTGAAAACCTCAAACGCGAAGACGAAGTATGCCCAGGCAGACTGTCAAGAGGTAGATTTGGAGATTGTAAACGAGGAAGAAAGCCAACAGCCGAGCCGGTGCCATAAAATTGCCATCCTCTCATTCAGAGAGGCCATTGTCAGAAACCAAATTAAGTCCCTGCTGGACAACTATGATTATGAGGCAGCTTTGCAGCTGGTCGCTAGTCAGAAGTCCTTTCGTAACGGTAAAGAGATCAGAAAGAAACTTAAGGAACTTATTGACGATATCAAAATGCACCGCGTTTTCAGTTATCTGATAAAGCAATACCCGCGTAATGAGAAACTTCAGAAGGCCCTTCTCCATACGATCCTGTTGGAGATGCGCCATCAGCGCGGCGATATAGCTGAGACATTAATTCGCGTTAAGTCAATCGCTGAGTACATTGTTGAGCAGTACATACAGAAGAATTACCCCTACCTCATTATTTACAAGGAAGATAAACCGTATTTTAATGTCTCGTATTCCCAGGAACTCACTGAATCGTACCTGGCGTTAATGGATTCCCGAAATAAGAAAACGAATAAGAAGATGACTGTAGATAGCCTCGACCGGATACTGGGATTCCCGGCCTATCGGGACTTTCTTCAGTTGCTCGAGGCGAGTAATGAAATGACAAATGAAATGAACAAGGTCAACGAGATCAACAATCTGCGGAACAAAGTGGCACACAATTTGGATAGCTTGAACCTCGACCGCGATAAGAACGGCCGTAAGATTACTAACGCAGTGACCGCTGTTCGTACTATGCTCCTTGCTGTGTTTCCTGAAGTGCAAGAGAATGATTTTCACTACCTCAAACAGTTCAACCAATCCATCAAGGAGCTGCTTTAATACTAGTAGCGGCCGCTGCAG

**Eicsm6 Composite part:**
GAATTCGCGGCCGCTTCTAGAGCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAAATGAAAATCCTGTTCAGTCCTATCGGTAATACTGACCCCTGGCGTAACGATCGTGACGGAGCAATGCTTCATATTGTCCGCCATTATCAGCCCGATCGGGTTGTTCTGTTCTTCACAGAATCTATATGGCAGGGTAACCAGCATTTTAGCGGGCAGCAGGCATTCGACTGGGTTAAGATTATACAATCTATAAATGAAAATTGCCAAATAGAGATAAAGTGCGACACGATCGAAGTCGAAAACGACTTTGACGCGTATAAGGACCTCTTTCACCAATATCTTGTTGAAGAGAAGCGCAAGTACCCAAACGCCGAGATATTTCTTAACGTAACCAGTGGTACCCCACAAATGGAGACGACGCTGTGTTTAGAGTACGTCACCTATCCAGATAAGATGCGGTGTATACAAGTGTCAACGCCACTCAAAACCTCGAACGCAAAGACGAAGTATGCACAGGCTGACTGTCAAGAGGTCGATTTGGAGATTGTGAACGAGGAAGAAAGCCAACAGCCGTCCCGTTGCCATAAAATTGCCATACTCTCCTTCCGAGAGGCGATTGTGCGCAACCAAATTAAGAGCCTCTTGGACAACTATGATTATGAGGCTGCATTGCAATTAGTGGCCTCTCAGAAATCCTTTCGGAACGGAAAAGAAATACGTAAGAAATTGAAGGAACTTATAGATGATATCAAAATGCACCGCGTCTTTTCCTATCTGATTAAGCAATACCCTCGGAATGAAAAGTTGCAGAAGGCGTTACTTCATACTATCCTGCTGGAGATGCGCCATCAGCGGGGCGATATAGCGGAGACCTTGATTCGCGTTAAATCCATAGCCGAGTACATTGTAGAGCAGTACATTCAGAAGAATTACCCGTACCTTATAATTTATAAAGAGGATAAACCATACTTCAATGTGTCGTATAGCCAGGAATTAACCGAGTCATACTTAGCCCTCATGGATTCGCGTAATAAGAAAACGAATAAGAAGATGACGGTCGATTCATTAGACCGCATTCTGGGTTTCCCTGCCTATCGTGACTTTCTGCAACTCCTGGAAGCAAGTAATGAAATGACGAATGAAATGAACAAGGTGAACGAGATAAACAATCTGCGGAACAAAGTAGCACATAACTTAGACAGTCTTAATTTAGATCGTGACAAGAATGGTCGCAAAATCACGAATGCGGTAACTGCGGTGCGCACCATGCTGTTGGCGGTCTTCCCAGAGGTTCAGGAAAACGACTTCCATTATTTAAAGCAATTTAATCAGAGCATTAAAGAACTTTTGCATCACCATCACCATCACTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAG

**DNaseI basic:**
GAATTCGCGGCCGCTTCTAGATGCGTGGAACAAGACTTATGGGATTACTCCTTGCGTTAGCAGGCCTTTTACAACTCGGGCTTAGTCTGAAAATTGCGGCATTTAATATTCGAACTTTCGGCGAAACGAAAATGTCAAACGCGACTTTAGCGTCCTATATAGTGCGAATAGTACGGCGCTATGATATTGTACTTATTCAAGAAGTACGTGATAGTCATTTGGTAGCCGTCGGCAAACTGCTTGATTACTTGAATCAAGACGATCCTAATACTTATCATTACGTTGTGAGCGAACCCTTAGGTCGTAATTCCTATAAGGAACGTTACCTTTTCTTGTTTCGTCCGAATAAAGTCTCGGTTTTGGATACGTATCAATATGATGATGGTTGTGAATCATGTGGTAATGATTCTTTTAGTCGCGAACCAGCCGTCGTGAAATTTTCTTCACATTCGACAAAAGTAAAAGAGTTCGCTATAGTGGCACTTCATTCTGCACCGTCCGATGCCGTCGCAGAAATCAACAGCCTGTATGACGTGTATCTCGACGTGCAACAAAAGTGGCATCTGAATGACGTTATGCTTATGGGAGACTTTAACGCCGATTGTTCCTATGTTACGTCAAGTCAATGGTCTAGTATACGTCTCCGGACCAGTAGCACATTTCAATGGTTAATCCCAGATTCCGCTGATACAACTGCGACCTCAACTAATTGTGCGTACGATCGCATTGTAGTTGCTGGAAGCCTTCTGCAATCTTCAGTAGTCCCAGGTTCAGCAGCCCCTTTCGACTTCCAAGCTGCATACGGACTGAGCAATGAGATGGCCCTGGCCATCAGTGACCATTACCCGGTGGAGGTGACGCTGACATAATACTAGTAGCGGCCGCTGCAG

**DNase1 composite:**
GAATTCGCGGCCGCTTCTAGAGCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAAATGCGGGGTACACGTTTGATGGGTTTACTCCTGGCTTTGGCCGGCTTGCTCCAATTGGGACTTAGTCTTAAAATTGCCGCGTTTAATATACGTACATTCGGCGAAACTAAAATGTCTAACGCCACATTGGCTTCTTATATCGTGCGTATTGTTAGACGATATGATATTGTGTTGATTCAAGAAGTTCGTGATAGTCATTTGGTTGCCGTCGGCAAACTTTTAGATTACCTTAATCAAGACGATCCGAATACATATCATTACGTTGTGTCCGAACCTTTGGGGAGAAATTCTTATAAAGAACGGTATCTTTTCCTGTTTCGACCGAATAAAGTTAGCGTACTCGATACTTATCAATATGATGATGGGTGTGAAAGCTGTGGCAATGATTCTTTTAGCCGTGAACCGGCAGTCGTAAAATTTAGTTCACATTCGACTAAAGTGAAAGAGTTCGCAATAGTCGCATTACATTCTGCACCGTCTGATGCTGTCGCGGAAATCAACTCGTTATATGACGTGTATTTAGACGTTCAACAGAAATGGCATTTAAATGACGTTATGCTGATGGGTGACTTTAACGCGGATTGTTCTTATGTCACGTCTTCTCAATGGTCTAGTATTAGACTTCGGACTTCAAGTACTTTTCAATGGTTAATCCCAGATAGCGCAGATACAACCGCCACCTCAACCAATTGTGCTTACGATCGGATAGTCGTTGCGGGTAGTCTCTTACAATCTAGCGTTGTCCCGGGATCAGCGGCCCCGTTCGATTTTCAGGCCGCTTATGGTTTGAGTAACGAAATGGCACTTGCAATTAGCGATCACTATCCCGTTGAAGTAACCTTGACCTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAG

**NisinPV Coding Sequence:**
GTGCAACCTTGGGTAGGCTTTACTTAGCTAAAGGCCTTAGGGGATGGAATTCGCGGCCGCTTCTAGATGTCGACCAAGGACTTCAATCTGGACCTTGTTAGCGTCAGTAAGAAGGACTCCGGAGCGTCTCCGCGGATAACGTCAATATCTCTTTGCACCCCTGGCTGCAAGACTGGCGCGCTCATGGGATGCAATATGAAGACTGCGACCTGCCACTGCCCGGTCCATGTCTCTAAGTAATACTAGTAGCGGCCGCTGCAGCATCGTTTACTTGACTGAAAAGGGGCCTTCTCAAATTGG

**P2 + GFP :**
ATTAAATACAAATTACATTTAACAGTTAAGTATTTATTTCCTACAGTTAGGCAATATAATGATAAAAGATTGTACTAAATCGTATAATGACAGTGAAAAGAGGAGAAAATGCGCAAGGGCGAGGAGTTGTTTACAGGCGTGGTTCCGATCTTGGTCGAGCTTGACGGAGACGTAAACGGTCATAAGTTCAGCGTGTCCGGGGAAGGCGAGGGCGACGCTACCTATGGCAAGTTAACGCTGAAGTTCATCTGTACGACCGGTAAGCTTCCGGTCCCCTGGCCTACTTTGGTTACAACCTTTGGATACGGGGTGCAGTGTTTCGCACGATATCCGGACCACATGAAGCAACACGATTTCTTTAAATCAGCAATGCCAGAGGGATACGTTCAAGAGCGCACCATTTTCTTTAAGGACGATGGCAATTATAAAACGAGAGCAGAGGTAAAATTCGAGGGAGACACGTTAGTGAACCGAATTGAATTGAAGGGAATCGACTTCAAAGAGGACGGCAATATCCTCGGCCATAAGTTAGAGTATAATTACAATAGTCATAACGTGTACATAATGGCTGATAAGCAGAAGAACGGCATTAAGGTAAACTTTAAGATCCGTCATAACATCGAGGACGGCTCGGTACAGCTGGCTGATCACTACCAGCAGAACACGCCTATCGGAGACGGACCGGTTTTGCTTCCTGATAATCACTATCTTTCTACCCAGAGTGCATTGTCAAAGGACCCTAATGAGAAACGCGATCATATGGTATTGTTGGAGTTCGTCACCGCAGCGGGCATAACTCACGGTATGGACGAGCTGTATAAGTAATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGCTTTCGAAGGCTTAGGCGGGAG

**AgrA + terminator:**
AAAGAGGAGAAATACTAGATGGAGATTGCCTTGGCGACTGACAACCCCTACGAAGTATTAGAGCAAGCGAAGAATATGAATGATATTGGTTGTTACTTCCTTGACATCCAATTATCCACAGATATAAATGGAATAAAGCTCGGAAGCGAGATACGCAAGCACGACCCAGTGGGCAATATCATCTTTGTCACGTCCCACTCCGAATTGACATACTTAACCTTTGTGTATAAAGTGGCCGCGATGGACTTTATATTCAAGGATGACCCTGCCGAATTGCGCACACGTATAATTGATTGCTTAGAGACAGCTCATACTCGGCTTCAGTTGCTTAGCAAGGACAATAGCGTTGAGACAATCGAGCTTAAGCGCGGGAGTAATTCGGTGTATGTGCAGTACGACGACATTATGTTCTTTGAGAGTTCTACAAAATCTCATCGCTTAATTGCACACCTTGACAACAGACAAATAGAATTTTACGGAAACCTTAAGGAATTATCACAACTGGACGACAGATTCTTTCGGTGCCACAATAGTTTTGTGGTGAACAGACATAATATTGAGAGCATTGATTCAAAGGAAAGAATAGTCTATTTTAAGAATAAAGAGCATTGCTACGCAAGCGTGCGGAACGTAAAGAAGATTTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTAT

**pBAD + AgrC:**
ACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCAAGATAGTCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCGTTTTTTTGGGCTAGCAAAGAGGAGAAAATGATCCTTATGTTTACAATCCCTGCGATCATCTCAGGAATCAAATATTCAAAATTGGACTACTTCTTTATTATTGTTATAAGTACGTTATCATTATTCCTCTTTAAGATGTTTGACAGTGCTTCTTTGATAATTCTCACCAGCTTTATTATTATTATGTACTTTGTGAAGATCAAGTGGTACTCGATTCTCCTGATAATGACAAGCCAAATAATTTTGTATTGCGCAAATTATATGTACATAGTAATTTATGCATATATTACTAAGATTTCTGACTCAATATTTGTCATTTTCCCAAGCTTCTTCGTAGTATACGTAACTATATCGATCTTATTCAGTTACATCATTAACCGGGTATTGAAGAAAATTTCCACACCCTATTTAATCTTAAATAAGGGGTTCTTGATTGTTATTTCAACAATATTGCTCTTGACGTTTAGCCTTTTCTTCTTCTATTCCCAAATAAATTCGGACGAAGCGAAGGTGATTAGGCAATATTCTTTTATTTTCATCGGCATAACAATTTTCCTCTCAATCCTCACGTTTGTGATTAGCCAATTTCTTCTGAAGGAGATGAAATATAAACGGAATCAGGAAGAGATTGAAACATACTACGAATATACGTTGAAAATAGAGGCTATTAATAATGAGATGCGGAAGTTCCGCCATGATTATGTCAATATCCTTACGACGCTTTCAGAGTATATTCGCGAGGATGATATGCCGGGATTACGCGACTACTTTAATAAGAATATAGTACCTATGAAGGACAATCTTCAGATGAATGCCATCAAATTAAATGGCATTGAGAATCTTAAGGTTCGGGAAATTAAAGGCTTGATAACAGCAAAGATTTTGAGAGCCCAAGAGATGAATATTCCGATCTCAATTGAGATTCCGGATGAGGTGAGCTCGATCAATTTAAATATGATAGATTTAAGCAGATCAATTGGGATTATTTTGGATAATGCAATTGAGGCGAGCACAGAGATTGATGATCCTATCATCCGTGTAGCGTTCATTGAATCGGAAAATTCGGTCACATTCATTGTCATGAATAAGTGTGCCGATGACATTCCTCGAATCCATGAGCTCTTTCAAGAGTCCTTTTCCACAAAAGGCGAGGGCAGAGGGCTCGGGTTGAGTACATTGAAGGAAATTGCAGACAATGCTGATAATGTGCTCTTGGATACAATAATTGAGAATGGCTTCTTCATCCAAAAGGTTGAGATTATTAACAATTAATAA

**Primers

+**

**Primers for normal PCR:**

* Amplification of biobrick part

  | Primer type | Sequence | % GC | Tm | Length |
  | --- | --- | --- | --- | --- |
  | Forward | 5’-gatggaattcgcggccgcttcta-3’ | 56.5 | 61.7 | 23 |
  | Reverse | 5’-gatgctgcagcggccgctactagta-3’ | 60 | 64.4 | 25 |
* Linearized plasmid backbone synthesis

  | Primer type | Sequence | % GC | Tm | Length |
  | --- | --- | --- | --- | --- |
  | Forward | 5’- gtgctgcagtccggcaaaaaa-3’ | 52.4 | 59.3 | 21 |
  | Reverse | 5’-gtgaattccagaaatcatccttagcg-3’ | 42 | 66 | 26 |
* Sequencing primers

  | Primer type | Sequence | % GC | Tm | Length |
  | --- | --- | --- | --- | --- |
  | Forward(VF2) | 5’-tgccacctgacgtctaagaa-3’ | 50 | 60 | 20 |
  | Reverse(VR) | 5’-attaccgcctttgagtgagc-3’ | 50 | 60 | 20 |

**Primers specific for Hi-Fi DNA assembly:**

* For Unidirectional Assembly

  |  |  |
  | --- | --- |
  | Pbad\_RBS\_AgrC\_fwd\_uni | 5’-tctggaattccaacattgattatttgcacgg-3’ |
  | Pbad\_RBS\_AgrC\_rev\_uni | 5’-tttctcctctttttattaattgttaataatctcaaccttttg-3’ |
  | RBS\_AgrA\_fwd\_uni | 5’ taacaattaataaaaagaggagaaatactagatg 3 |
  | RBS\_AgrA\_rev \_uni | 5’ tttgtatttaattataaacgcagaaaggcc 3 |
  | P2\_GFP\_fwd\_uni | 5’ttctgcgtttataattaaatacaaattacatttaacagttaagtatttatttcctacagttag 3’ |
  | P2\_GFP\_rev\_uni | 5’ ggactgcagcacctcccgcctaagccttcg 3’ |
  | PSB1C3\_fwd\_uni | 5’ gcttaggcgggaggtgctgcagtccggcaaaaaag 3’ |
  | PSB1C3\_rev\_uni | 5’ aaataatcaatgttggaattccagaaatcatccttagcg 3’ |
* For Bidirectional Assembly

  |  |  |
  | --- | --- |
  | Pbad\_RBS\_AgrC\_fwd\_bi | 5’- tttgtatttaatacattgattatttgcacgg-3’ |
  | Pbad\_RBS\_AgrC\_rev\_bi | 5’-tttctcctctttttattaattgttaataatctcaaccttttg-3’ |
  | RBS\_AgrA\_fwd\_bi | 5’ taacaattaataaaaagaggagaaatactagatg 3 |
  | RBS\_AgrA\_rev \_bi | 5’ ggactgcagcactataaacgcagaaaggcc 3 |
  | P2\_GFP\_fwd\_bi | 5tctggaattccactcccgcctaagccttcg 3’ |
  | P2\_GFP\_rev\_bi | 5’aaataatcaatgtattaaatacaaattacatttaacagttaagtatttatttcctacagttag 3’ |
  | PSB1C3\_fwd\_bi | 5’ ttctgcgtttatagtgctgcagtccggcaaaaaag3’ |
  | PSB1C3\_rev\_uni | 5’ gcttaggcgggagtggaattccagaaatcatccttagcg 3’ |

**Microbial strain

+**

**E. coli DH5α:** This strain is used for only cloning and assembly of different gene fragments.

**E. coli BL21:** This strain is used for study expressions.

**Pseudomonas aeruginosa:** This strain is used for performing the Biofilm assay.

**Media Preparation

+**

<https://benchling.com/s/prt-dUYnUJLsuWZWQ1NQGeR8>

**Stock Solution Preparation

+**

**0.5 M EDTA stock solutions**

For preparation of 30ml stock solution

* Weigh out 5.583 grams of EDTA disodium salt.
* Dissolve in 25ml of milli Q water and adjust pH with solid NaOH to reach the pH of 8
* Add 5ml milli Q water to make the total volume to 30ml
* Autoclave for 15mins at 121 °C

**50X TAE Buffer**

For preparation of 250ml solution

* Weigh out 60.5g Tris base and dissolve approximately in 200ml milli Q water
* Carefully add 14.275ml of 100% glacial acetic acid
* Add 25ml of 0.5 M EDTA (pH 8)
* The pH of buffer should be around 8.5
* Store at room temperature

**10X TAE Buffer working solution**

Add 20ml of 50X TAE buffer to 1000ml of milliQ water.

**1M NaOH preparation for pH adjustments**

To prepare 20ml of 1M NaOH add 0.8g of NaOH pellets in 20ml of dH2O. Then store it in a falcon tube.

**Crystal Violet solution preparation**

For 1L solution

1. Add 1g of crystal violet to 1000ml of dH2O and then do the magnetic stirring for solubilising.

**Antibiotics Stock and working solutions**

As per the requirement, the stock and working concentrations can be made by referring to the corresponding concentrations provided in the table below.

[Download AntibioticsConcentrations.csv](https://static.igem.org/mediawiki/2021/2/21/T--IISER_Kolkata--file-Experiment-AntibioticsConcentrations.csv)

**Stock for Primers**

* Check the concentrations of each primer provided by the vendor
* Need to make the stock concentration of 100pm/μl
  + Centrifuge the empty vials at 30,000 rpm for 30 secs.
  + Heat the NFW at 95°C for 10mins then at 55°C for 30 mins
  + Pour the NFW to the mentioned volume to make the stock concentration (100pm/μl) in each tube of primer. Remember to perform this step inside the hood.
* Leave the tubes in 4°C for 4-5 hrs.
* Take it out from 4°C and mix by soft pipetting or light vortex. Then store it at -20°C.
* For making a working concentration of 10pm/μl add 10μl of stock solution to 90 μl of NFW.

**Resuspension of biobricks from iGEM kit Plates**

* Label the plates properly and carefully with a marker (for reference check the well contents from iGEM website)
* Take 10μl of NFW using a micropipette. Punch a hole in the aluminum foil for the well you want to resuspend with the tip of the micropipette tip.
* Slowly release the water into the well.
* Gently pipette inside the well for a few times. The solution will be red due to the presence of cresol dye.
* Keep the mixture there for 5 mins
* Now transfer it to a PCR tube and store it in -20°C. Label it properly before storing

**Resuspension of DNA fragments ordered from Twist Bioscience -**

* Briefly centrifuge the tube and resuspend the DNA in NF-tris EDTA (TE Buffer), pH 8 or 10mM Tris HCl, pH 8 to the desired concentration.
* A concentration of 10ng/μl is recommended for a stock solution.
* Prepare aliquots of the stock and make working aliquots.
* For long-term storage, freeze DNA at -20°C or -80°C

**DNaseI stock preparation**

Lyophilized form of Bovine DNaseI was obtained from sigma aldrich (D2025-15KU). Resuspension and stock preparation was done as per the guidelines from the company:

1. Lyophilized form which contains approximately 5 mg of DNaseI was resuspended in 0.15M NaCl solution making up to a concentration of 5mg/ml.
2. Working concentrations were made by diluting it further in 0.15M NaCl or Media directly.
3. Stock solution was stored in -20 ℃ to prevent degradation.

**Competent Cells Preparation**

**Introduction**

Preparing competent cells of Ecoli KRX,BL21

**Materials**

**TFB-1(pH-5.1) for 30 ml**

> 10 mM RbCl - 0.36 g
> 50 mM MnCl2 - 0.19 g
> 30 mM CH3COOH+ - 0.08 g
> 100 mM CaCl2 - 0.03 g
> 15 % glycerol - 4.5 ml

**TFB-2(pH-6.8) for 15 ml**

> 10 mM MOPS -0.031 g
> 10 mM RbCl-0.012 g
> 15 mM CaCl2-0.124 g
> 15 % glycerol-2.25 ml

**Procedure**

**STEPS**

1. Pick a single colony of cell and inoculate in 10 ml of LB
2. Grow it overnight at 37 ° C overnight by shaking
3. Add 1 ml of overnight grown culture to 100 ml of pre warmed LB broth in 500 ml flask
4. Shake on and grow at 37 ° C till the OD reached 0.5
5. Cool the culture on ice for 5 min and then transfer culture to round bottom centrifuge tube(Falcon)
6. Collect the cells by centrifugation(5 min,4000g) at 4° C
7. Discard the supernatant and keep the cells on ice
8. Resuspend the cells on a cold TFB-1 buffer(30 ml) and inoculate on ice for 90 min.
9. Collect the cells by centrifugation(5 min,4000g, 4 ° C)
10. Discard the supernatant and keep cells on ice.
11. Resuspend the cells in 4 ml of ice cold TFB-2.
12. Prepared 50 to 100 ml aliquots in 1.5 ml centrifuge tube
13. keep all those tubes in -80°C.

**Cloning’s Method**

**Plasmid Miniprep

+**

<https://benchling.com/s/prt-jXq3KGdv9zF9pV3Rdu5U>

**PCR Amplification

+**

<https://benchling.com/s/prt-IYttDOXR7v1ti5zjxV7I>

**PCR Cleanup

+**

<https://benchling.com/s/prt-HuCXRJMMWe891Ci1SltV>

**Gel Extraction

+**

<https://benchling.com/s/prt-aaec85DE3RcBg4iQxLHz>

**Double Digestion

+**

<https://benchling.com/s/prt-7zPmsYI7dKKEdr16lH3X>

**Gel electrophoresis

+**

<https://benchling.com/s/prt-8N5qTzyZoyqNgIGCZs7a>

**Ligation

+**

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. The molar ratio of insert: plasmid is 3:1 in the below table .) We used [NEBioCalculator](http://nebiocalculator.neb.com/#!/) to calculate molar ratios.

   The table below is given for 20ul Reaction

   | Competent |  |
   | --- | --- |
   | T4 DNA Ligase Buffer (10X)(Should be thawed and resuspended in room temperature) | 2 μl |
   | Vector DNA (4 kb) | 50 ng (0.020 pmol) |
   | Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
   | Nuclease-free water | to 20 μl |
   | T4 DNA Ligase | 1ul |
2. The reaction is gently mixed by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, it is incubated at 16°C overnight.
4. For blunt ends or single base overhangs, it is incubated at 16°C overnight.
5. The mixture is heat-inactivated at 65°C for 10 minutes.
6. The mixture is chilled on ice and transformed 1-5 μl of the reaction into 50 μl competent cells.

**Colony pcr

+**

<https://benchling.com/s/prt-J42Df7DdnlCdRDWq6ofr>

**HiFi DNA Assembly**

We have assembled 4 fragments (including plasmid backbone) using NEB HiFi DNA Assembly kit. We have calculated the numbers of picomoles of each fragment using the [NEBiocalculator](https://international.neb.com/external-links/nebiocalculator).

**Set Up the reaction mixture of 50 microliter**

|  |  |
| --- | --- |
| Recommended DNA Molar Ratio | vector:insert = 1:1 |
| Total Amount of Fragments | 0.2–0.5 pmols\*\* X μl |
| NEBuilder HiFi DNA Assembly Master Mix | 10 μl |
| Deionized H2O | 10-X μl |
| Total Volume | 20 μl |

**Transformation Protocol**

**Introduction**

Transfer of miniprep plasmids or Ligation mixture into Escherichia coli KRX or BL21

**Materials**

* Ice
* Competent cells
* Ligation mixture or miniprep plasmids
* SOC media
* Hot water bath
* Incubator
* Timer
* Agar plates
* cell spreader
* Bunsen burner
* Laminar air flow hood

**Procedure**

The following steps are to be followed in order -

1. Take out the competent cells (100μl/50μl) from -80°C storage and place in ice.
2. Add ligation mixture (10μl) or miniprep Plasmids (1 or 2 μl) to the 100μl competent cells by pipetting. While pipetting make sure that the tip of the micropipette tips does not come in contact with the competent cells. Close the tube and mix them by gently tapping on the side of the tube.
3. Keep the tube in ice from 30 mins.
4. In the meantime switch on the hot water bath and set the temperature to 42°C and also keep the SOC media in the incubator at 37 °C.
5. Place the tube in a floating rack and then heat shock it for 45 seconds at 42°C, use a timer for accurate measurements of time.
6. Now put the tube immediately into ice and keep it for 5 mins.
7. Then add SOC media to make up the volume to 1000μl and incubate it for 1.5 hours in shaking condition at 37°C.
8. In the meantime keep the agar plates at room temperature.
9. Post incubation takes 100 μl from the tube and then spreads it with a cell spreader. Then take the rest of the contents of the tube and centrifuge it at 10,000 x g for 3 mins, pour out the maximum supernatant and then mix the cell pellets with the leftover supernatant with a pipette and again spread it on another agar plate. Store the plate in an incubator at 37°C for 12-16 hours.

**Quantitative Assays**

**Biofilm Assay

+**

Initially method of biofilm formation in 96 well plate was standardised by using different choices of media ,incubation conditions ,dilutions and incubation times.The following is the optimised protocol for maximum yield of biofilm:

<https://benchling.com/s/prt-YNCdyxyfi1AgPzUeXA98>

**DNase1 Assay

+**

The effect of DNaseI on biofilm of Pseudomonas aeruginosa is studied using 96 well microtiter experiments. To optimize the concentration of DNaseI required for treatment, the effect of a gradient of DNase I in the range of 0–50 µg/mL was studied. As a negative control for the experiment, uninoculated wells containing media and diluent were treated similarly and readings obtained were subtracted from the test readings. Positive control wells containing inoculated media without DNaseI were considered as “Control A595 nm”.The biofilm percentage reduction is then calculated as:

$$ BPR = \left( \frac{Control \ A595 nm - test \ A595 nm}{Control \ A595 nm} \right) \times 100 $$

Inorder to determine the optimum concentration and time for DNase I treatment,we did assay at two levels:

1. **Pretreatment**

   It was done to determine the optimum concentration of DNaseI which gives maximum degradation.

   * A serial dilution of DNaseI (0-25 µg/ml) was prepared in NB media.
   * Primary cultures were directly diluted to this media and added to 96 well plates as per the procedure mentioned in above protocol.
   * The layout used is as follows:

     ![](https://static.igem.org/mediawiki/2021/f/ff/T--IISER_Kolkata--image-Experiments-BioFilmlayout.png)
   * Biofilm was allowed to develop in the presence of DNase1 for 72 hrs.
   * Plates were washed and processed as per the procedure mentioned in above protocol.
   * Absorbance was measured at 595 nm to quantify biofilm.
   * Biofilm percentage reduction at different concentrations were compared and analysed.
2. **Posttreatment**

   This was done to determine the optimum time required for maximum biofil degradation for the optimal concentration determined by pretreatment.

   * Pseudomonas biofilm was allowed to develop in 96 well plates for 72 Hrs as per the protocol mentioned before.
   * Well constituents,particularly bacteria, were then washed off in dH20.
   * Media containing optimum concentration determined by pretreatment was then added into each well and incubated for different times ranging from 0-50 min.
   * Plates were further processed by adding Crystal violet followed by Acetic acid.
   * Biofilm quantification was carried out at 595nm.
   * Biofilm percentage reduction was calculated and further analysed.

**Fluorescence Assay

+**

* One LB agar plate containing bacteria with a unidirectional circuit and another LB agar plate with a bidirectional circuit containing bacteria were obtained.
* Primary cultures of both the bacteria were given separately in 10ml of LB agar containing Chloramphenicol.
* The culture was incubated overnight at 37 degrees celsius.
* 25ml of fresh LB media (Chl+) was added in 250 ml autoclaved conical flasks.
* Half of the flasks were inoculated with 1ml of the primary culture of the unidirectional circuit containing bacteria each, while the other half were inoculated with 1ml of primary culture each of bidirectional circuit containing bacteria.
* The flasks were incubated at 37 degrees celsius till 0.6 OD (600nm) was reached.
* Flasks were induced with different concentrations of Aip molecules.
* To check the fluorescence, Polystyrene Nunc black 96-well fluorescence plate was obtained.
* 100ul of samples from each flask was added to the 96-well plate in triplicates. 100ul of LB media was added to each well to dilute the samples.
* 200ul of LB media was added to three wells as a negative control.
* The fluorescence was measured in a plate reader, where excitation wavelength was set at 395nm and fluorescence wavelength was measured at 509nm.
* All the flasks were incubated at 30 degrees celsius to allow protein expression.
* The fluorescence was measured at different time points.

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# Team:IISER Kolkata/Results

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* Overview
* DNaseI biofilm assay
* The AIP producer
* The improved AIP sensor
* Characterization of LwaCas13a

**Overview**

Due to unexpected changes in plan during COVID-19,our team arrived in campus to access lab facilities only by August.It took another one month for us to procure all requirements.So,we as a team focussed primarily on the treatment part of our project as it was the best option to yield successful results within the limited amount of time that we got for lab work .

Developing an antibiotic-free system for curing bovine mastitis was accomplished by incorporating a combination of Nisin PV and DNaseI under the regulation of AIP-I regulated quorum-sensing unit consisting of AgrC-AgrA signaling components.Inorder to develop of therapeutic system to its final form,we have to estimate and characterize each component in the genetic circuit.

With proper planning and hardwork,,we successfully cloned and characterized the following proteins involved in our antibiotic-free treatment method within a time span of one month.All these results were accompanied with a series of trial and error and troubleshooting as well.

**DNaseI biofilm assay**

Our aim is to genetically modify E Coli to produce bovine pancreatic DNaseI.The use of DNaseI as a molecule to degrade bacterial biofilm and hence favor the activity of antimicrobial peptides is well established in the literature.It particularly targets the extracellular matrix DNA in biofilm hence destabilizing it and exposing the bacteria to external factors.Here we intended to capture this role of DNaseI by showing its biofilm degrading activity.

**Cloning and expression**

Initially, gene fragment of DNaseI coding sequence with biobrick prefix and suffix which was synthetically obtained from Twistbioscience was amplified using Routine PCR using biobrick specific primers. This was then ligated into linearized plasmid backbone of PSB1C3 after restriction digestion by EcoRI and PstI following normal biobrick assembly.E Coli DH5ɑ was transformed and further verified using colony PCR using sequencing primers.The size of gene fragment after amplifying with sequencing primer was simulated as 1161 bp using Snapgene insilico PCR simulator.

![BBa_K3799002--cPCR.jpeg](https://static.igem.org/mediawiki/parts/5/5f/BBa_K3799002--cPCR.jpeg)

Colony PCR result of DnaseI coding sequence.A size of 1161 bp was calculated using Snapgene insilico PCR

For expressing the protein in Ecoli BL21, we cloned a composite part of DNaseI which consists of IPTG inducible Plac promoter(R0010) along with strong RBS(B0034) and double terminator(B0015). This part was obtained synthetically from Twistbioscience. Gene fragment was amplified by Routine PCR using biobrick specific primers and then ligated to linearized plasmid backbone of PSB1C3 following normal biobrick assembly. Double digestion by EcoRI and PstI was done using optimized protocols for double digestion. Ecoli DH5ɑ was initially transformed and transformants were screened and verified using colony PCR using sequencing primers. Snapgene in silicon PCR simulator was used to determine the size of gene fragments after colony PCR.

![IISER_Kolkata--composite.jpeg](https://static.igem.org/mediawiki/parts/f/f0/IISER_Kolkata--composite.jpeg)

![IISER_Kolkata--cPCR-DNASE_COMP.jpeg](https://static.igem.org/mediawiki/parts/d/d7/IISER_Kolkata--cPCR-DNASE_COMP.jpeg)

Colony PCR result of DnaseI composite part gave succesful result.A size of 1512 bp was calculated using Snapgene insilico PCR

Further,we extracted the plasmid from transformed colonies and transformed Ecoli BL21 for expressing protein.However due to COVID-19 restrictions,we did not get enough time to achieve our complete goaluptoprotein purification.So for further characterization,we used industrially prepared bovine pancreatic DNaseI(D2025).

**Characterization**

1. **Standardisation of biofilm assay**
   Characterization of DNaseI was carried out by performing the standard Biofilm assay in 96 well plate as per previously established protocols.As Pseudomonas aeruginosa was easily available in our laboratory and protocols for developing their biofilm was previously well established,we choose to use them for growing biofilm.We had to standardize the protocol by varying experimental conditions at determine the conditions for maximum yield of biofilm before starting DNaseI treatment.We varied media type,dilution and days of incubation in our experiments.

   From a literature survey,we found a number of media that suits for biofilm growth which includes Luria broth(LB),Nutrient broth(NB) and Tryptone soytone broth(TSB).We initially grew bacterial biofilm without DNase in different media for different days of incubations(24 hrs and 72 hrs) to determine the incubation time for maximum growth of biofilm in 96 well plates at 37 ℃.It was observed that biofilm yield after 24 hrs of incubation is 84.3% lesser in LB,89.2% lesser in NB and 87.6 % lesser in TSB media.Also biofilm yieldin NB media was maximum showing a variation of 44.4% higher than LB and and 59.5% higher than TSB.

   ![IISER_Kolkata--day.jpeg](https://static.igem.org/mediawiki/parts/b/be/IISER_Kolkata--day.jpeg)

   Biofilm quantification for varying media and incubation time

   We also studied the variation in biofilm yield for two different dilutions of secondary culture provided in 96 well plate.Optical density of primary culture was diluted to 0.1 and 0.05 OD in fresh medium before adding to 96 well plate.This was then incubated for optimum incubation time of 72 hours as per previous experiment.Varying media was used here to serif there is any combined effect of media choice and dilution on biofilm yield.It was observed that at 0.05 OD,biofilm yield was about 45 % lesser in LB media,50.4% lesser in NB media and 50.1% lesser in TSB media.Also,NB media gave about 38.3% and 59.3 % higher yield of biofilm compared to LB and TSB media respectively.

   ![IISER_Kolkata--dilution.jpeg](https://static.igem.org/mediawiki/parts/9/92/IISER_Kolkata--dilution.jpeg)

   Biofilm quantification for varying media and dilution

   To sum up,we standardised the biofilm assay in 96 well plate by choosing NB media for bacterial growth,dilution of 0.1 and 72 hr incubation for maximum yield of biofilm.
2. **DNaseI -Biofilm assay**
   Next,we used the standardardised protocol for developing pseudomonas biofilm and checking the effect of DNase I on it.DNaseI activity was assessed in two stages to determine the optimum incubation time and concentration of DNaseI required to attain maximum degradation of bacterial biofilm.Degradation rate was estimated by comparing it in terms of Biofilm percentage reduction(BPR) which is given by:

   $$ BPR = \left( \frac{Control \ A595 nm - test \ A595 nm}{Control \ A595 nm} \right) \times 100 $$
   * **Pre treatment of bacterial biofilm with DNaseI**
     Here,we pre incubated developing bacterial biofilm with different concentrations of DNaseI for 72 hrs.Absorbance of biofilm solution was calculated following crystal violet treatment and BPR calculated.It was observed that a concentration of 10 µg/ml gave maximum degradation.BPR did not further increase with increasing concentration.

     ![BBa_K3799003--pre.jpeg](https://static.igem.org/mediawiki/parts/f/f0/BBa_K3799003--pre.jpeg)

     Biofilm percentage reduction under varying concentration of DNaseI
   * **Post treatment of predeveloped biofilm with DNaseI**
     Bacterial Biofilm was allowed to to develop for 72 hrs in 96 well plate.Bacterial colonies were removed and then further treated with media containing 10 µg/ml of DNaseI.It was incubated for different time points and Biofilm percentage reduction was determined after measuring absorbance.After 40 min of incubation,BPR was recorded to be maximum.

     ![BBa_K3799003--post.jpeg](https://static.igem.org/mediawiki/parts/4/4c/BBa_K3799003--post.jpeg)

     Biofilm percentage reduction by DNaseI for different incubation time

**Discussion**

DNaseI can effectively eradicate bacterial biofilm and hence increase susceptibility of bacteria to external agents.

**The AIP producer**

The quorum sensing molecule of S aureus ,AIP-1 regulates the activity of P2 and P3 promoter.We wanted to produce AIP-1 to characterize and study the activity of the improved AIP sensor that we created.

**Cloning and characterization**

For this we cloned the AIP-1 producer( I746001) with IPTG inducible pLac promoter(R0010).The plasmids for each of the genes were obtained from iGEM kit and amplified separately in DH5α cells.It was further digested following normal biobrick assembly.

![IISER_Kolkata--digestedplac.jpg](https://static.igem.org/mediawiki/parts/b/ba/IISER_Kolkata--digestedplac.jpg)

Double Digested Plac + plasmid backbone, and double digested agrBD

The digested DNA fragments were purified , ligated and transformed into E.Coli DH5α Colony PCR was performed to identify the colony containing our desired gene circuit.

![IISER_Kolkata--plac_agrbd_colonypcr02.jpg](https://static.igem.org/mediawiki/parts/b/ba/IISER_Kolkata--plac_agrbd_colonypcr02.jpg)

Colony PCR of insert(plac+agrBD)

Plasmid from desired colonies were further isolated and transformed to BL21 cells for expressing.Transformed BL21 was cultured in LB media at 37℃ till 0.6 OD (600 nm) was reached. Protein expression was induced with IPTG. Protein expression was allowed for 10 hours at 30℃. The bacterial cells were pellet down and the supernatant containing AIP molecule was filter-sterilised. The supernatant was lyophilised to increase the concentration of the protein content.

**Characterization**

The presence of AIP-I in transformants were verified by checking GFP intensity produced by P2 promoter in the AIP inducible GFP reporter gene circuit. P2 promoter is initially activated by AgrA which requires phosphorylation from AgrC.AgrC is activated in the presence of AIP-I.

**Verifying presence of protein**

Initially,E.Coli BL21 expression vector containing plasmid having AIP receiver- agrA and agrC system downstream to the pBAD promoter, and GFP under the AIP inducible P2 promoter was obtained. These bacterial cells were cultured in 2 different conical flasks containing LB media with Chloramphenicol. The culture was incubated at 37 ℃ till the OD 0.6 (600 nm). The AIP molecules were resuspended by adding nuclease-free water. AIP-I was given in one flask, while the AIP-I molecule was not added in the second flask. The GFP expression was quantified by measuring the fluorescence of the GFP using a fluorometer at time zero. Further, the samples were incubated at 30℃ for another 5 hours. Then the fluorescence of the sample was checked for every hour from hour 05 to 10 hours.

![IISER_Kolkata--Agrca-Intensity_vs_time.jpg](https://static.igem.org/mediawiki/parts/d/dd/IISER_Kolkata--Agrca-Intensity_vs_time.jpg)

GFP intensity in the presence and absence of AIP-I

The graphical representation of the data makes it clear that there is a significant difference in the fluorescence intensity observed in the AIP inducible sample as compared to the sample without AIP. It shows that our Protein genetic circuit has successfully expressed the AIP protein and the expressed protein is active.

**Concentration-dependent activity**

Further ,we also validated the change in expression of GFP reporter construct in response to varying AIP-I concentration.

Here,Ecoli BL21 cells expressing AIP sensor with reporter construct were incubated with AIP molecules which were resuspended by adding nuclease-free water. Different volumes of the resuspended AIP molecules were added to different conical flasks to induce protein expression. The flasks were incubated at 30℃ for 5 hours. The GFP expression was quantified by measuring the fluorescence of the GFP using a fluorometer. The obtained data were analysed to obtain the AIP concentration-dependent GFP expression.

![IISER_Kolkata--Agrca-Intensity_vs_conc.jpg](https://static.igem.org/mediawiki/parts/7/77/IISER_Kolkata--Agrca-Intensity_vs_conc.jpg)

GFP intensity with varying concentration of AIP-I

The analysis of the fluorescence data shows that the expression of GFP has increased with the addition of a higher volume of resuspended AIP molecules. That is, the GFP expression has increased with an increase in the AIP-I concentration in the media. This further proves that our cloned genetic circuit was able to successfully express the AIP-I molecule and release it in media in its active form.

The fluorescence intensity has dropped in presence of a very high concentration of AIP. This can be due to toxicity caused due to the high concentration of AIP.

**The improved AIP sensor**

Finally, we improved the AIP-1 sensor from S.aureus by accommodating the problem of leaky gene expression.The orientation of promoters are placed opposite which will prevent possible leaky gene expression.

**Introduction**

We observed that there is leaky gene expression in the Unidirectional circuit. We modified the existing part to reduce the leaky gene expression. By flipping the gene we expect that the leaky gene expression will be reduced. To check our hypotheses we have compared the expression of GFP in the unidirectional and the bidirectional genetic circuit.

**Cloning and expression**

Cloning of this gene circuit was done using NEB HI-FI DNA assembly.First, all the three gene fragments (pBAD-agrC, P2-GFP,agrA,) were extended by routine PCR using specific primers designed accordingly to give unidirectional circuit in which P2 and pBAD are oriented in same direction.PCR extension was verified by Gel electrophoresis and all inserts were correctly amplified(fig- Extended pBAD-agrC ~ 1.4 Kb, Extended P2-GFP ~ 1Kb, Extended agrA ~ 800bp and extended backbone~2kb).

![IISER_Kolkata--uni_colony_pcr.jpg](https://static.igem.org/mediawiki/parts/4/45/T--IISER_Kolkata--uni_colony_pcr.jpg)

PCR extension of pBAD-agrC,P2-GFP,agrA for unidirectional assembly

A linearized plasmid backbone of PSB1C3 was also extended using specific primers(extended primer~2.1kb).

![IISER_Kolkata--psb1c3.jpeg](https://static.igem.org/mediawiki/parts/6/6d/T--IISER_Kolkata--psb1c3.jpeg)

PCR extension of psb1C3 for unidirectional assembly

After that, the same four segments (pBAD-agrC, P2-GFP, agrA, and psb1C3 plasmid backbone) were PCR amplified with different sets of specific primers. This extended overlap helps flip the P2-GFP fragments. From the gel electrophoresis image below. We can observe similar band sizes as above because the difference in length of the extension is very low.

![IISER_Kolkata--bidi_extension.jpg](https://static.igem.org/mediawiki/parts/4/44/T--IISER_Kolkata--bidi_extension.jpg)

PCR extension of pBAD-agrC,P2-GFP,agrA for bidirectional assembly

![IISER_Kolkata--AgrA_extension.jpg](https://static.igem.org/mediawiki/parts/9/9a/T--IISER_Kolkata--AgrA_extension.jpg)

PCR extention of agrA segment

After the successful extension of all the segments, those extended fragments were assembled using Hi-Fi DNA assembly master mix. The reaction mixture was transformed in the E.Coil DH5a. After that, colony PCR of both the unidirectional and bidirectional circuit was performed using VF2 and VR primers to identify the bacterial colony with our desired gene circuits.

As evident from the colony PCR results, positive inserts for both unidirectional and bidirectional assemblies were obtained.

![IISER_Kolkata--uni_pcr_colony.jpg](https://static.igem.org/mediawiki/parts/0/0e/T--IISER_Kolkata--uni_pcr_colony.jpg)

Colony PCR result of unidirectional assembly with VF2 and VR

![IISER_Kolkata--bidicolonypcr.jpg](https://static.igem.org/mediawiki/parts/0/09/T--IISER_Kolkata--bidicolonypcr.jpg)

Colony PCR result of bidirectional assembly with VF2 and VR

Colony PCR yielded successful inserts for both Unidirectional and bidirectional assemblies.The plasmid was further transformed in Ecoli BL21 to express the desired proteins.

**Characterization**

After the successful assembling of both the fragments, we planned to see the expression level of GFP in both bidirectional and unidirectional circuits. For that, first, the AIP-1 generator (BBa\_K3799006) was expressed in 40ml of bacterial culture to obtain AIP-1 molecules. After expressing for 8-10 hours, the culture was centrifuged and the supernatant was filter-sterilized. The supernatant was lyophilized to concentrate the AIP-1 molecule. After full lyophilization, it was resuspended in 3 ml of NFW to make a stock solution of AIP1.

The stock solution of AIP-1 molecules was diluted to make solutions of different concentrations, from 0% to 100%, where 0% was the negative control and 100% was the one-third dilution of our stock solution.

The two types of cells were incubated (one with unidirectional plasmid and the other with bidirectional plasmid) at 30℃ for 8 hours without AIP-1.

The fluorescence intensity of those two types of cells in absence of AIP-1 molecules was measured at different time points and it was observed that there is a higher fluorescence intensity in cells with unidirectional circuits as compared to the cells with bidirectional circuits. This infers that the bidirectional circuit has less leaky gene expression than that of the unidirectional circuit.

![IISER_Kolkata--Bi-Intensity_vs_time.jpeg](https://static.igem.org/mediawiki/parts/0/07/IISER_Kolkata--Bi-Intensity_vs_time.jpeg)

Fluorescence assay of bidirectional and unidirectional circuits without AIP-1 vs Time

We also wanted to check whether this difference in leaky gene expression is consistent with different concentrations of AIP-1.

To check this, we induced our two types of cells (one with unidirectional plasmid and the other with bidirectional plasmid) with different concentrations of the AIP-1 solution that we mentioned above. From our data, we infer that this leaky gene expression of the unidirectional circuit is consistent in different concentrations of AIP-1 molecules. Also, we can observe a slight decrease in the gap of expression of GFP as the concentration of AIP-1 increases. This may be because of the fact that the ratio of leaky gene expression and the AIP-1 induced expression decreases as the AIP-1 concentration increases.

![IISER_Kolkata--Bi-Intensity_vs_conc.jpeg](https://static.igem.org/mediawiki/parts/9/93/IISER_Kolkata--Bi-Intensity_vs_conc.jpeg)

Fluorescence assay of bidirectional and unidirectional circuits at different concentrations of AIP-1 at 5hrs

**Discussion**

Our improved part part [BBa\_K3799057](http://parts.igem.org/Part%3ABBa_K3799057) indeed prevents leaky gene expression present in [BBa\_K1022100](http://parts.igem.org/Part%3ABBa_K1022100).

**Characterization of LwaCas13a**

In addition to what we achieved through our lab,we also made excellent collaborations with fellow teams to achieve our goals. For standardizing our detection part,we collaborated with iGEM GO Paris-Saclay for characterizing LwaCas13a protein with our miRNA. Cas13a protein is coupled with its crRNA and tested for its cleavage efficiency by activating it with bta-miR-7863 along with a ssRNA reporter to give a fluorometric output . The miRNA hybridizes with the crRNA and then activates the Cas13a RNase activity allowing it to cleave the reporter, emits fluorescence. Thus, it is possible to follow the detection of the miRNA in a quantitative way by measuring the fluorescence level.

![](https://static.igem.org/mediawiki/parts/4/40/IGEMGOPSCas13graph2.jpeg)

FIGURE 1 : Cas13a (cr/miR-7863) only activity with different concentrations of crRNAs and times of pre-incubation but constant miRNA concentrations 10 min of incubation (A), 30 min (B), 60 min (C), 120 min (D)

![](https://static.igem.org/mediawiki/parts/c/cc/IGEMGOPSCas13graph2iv.jpeg)

FIGURE 2 : Initiale velocities corresponding to the results presented on the Figure 7C Initial velocities (x = a.u./min)

**Discussion**

Based on the graphs, the optimal conditions for miR-7863 detection with Cas13a appear to be a crRNA concentration of 5nM and a 60-minute pre-incubation . Indeed, although a high fluorescent signal is observed with a 120-minutes pre-incubation, the corresponding profile at the other concentrations suggests that this is a result due to measurement or handling error. On the other hand, we can see that a pre-incubation of 10 minutes over 30 minutes seems to result in a low level of fluorescence, even for the most optimal crRNA concentrations

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