iGEM 2024 — Team iiser-kolkata

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{% block title %}Notebook{% endblock %}
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![Inclusivity Banner](https://static.igem.wiki/teams/5425/page-elements/notebook-66faf2ca9ffc7.webp)

# notebook

# Lab Experiments

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![Cycle Image](https://static.igem.wiki/teams/5425/page-elements/layout/design-3-66fb0bab07c60.webp)

◀

## Learn

▶

## Iteration 1: Circuit Designing

The math modellers found that the AiiA degradation tag (dtag) could help produce a stronger oscillation, hence is incorporated into the circuit. AiiA then lives just long enough for one round of oscillation, and doesn’t degrade AHL signal when the signal is actually needed, in the start of the next round of oscillation.
![](https://static.igem.wiki/teams/5425/mmm.jpg)
Finally, the circuit was finalised as follows.
![](https://static.igem.wiki/teams/5425/drylab/qs-circuit.webp)

## Iteration 2: Cell Cycle Arrest (CCA)

1. The RNA-RNA Docking showed a directly linear relationship between length of antisense mRNA and the spontaneity (given by the drop in Gibb’s free energy) of the steric blocking.
![](https://static.igem.wiki/teams/5425/ll/rna1.jpg)
![](https://static.igem.wiki/teams/5425/ll/rna2.jpg)

![](https://static.igem.wiki/teams/5425/ll/rna3.jpg)

Fig: Sense-Antisense mRNA Steric Blocking was deemed feasible through a molecular docking simulation.

![](https://static.igem.wiki/teams/5425/drylab/fitted-rna-rna.webp)

![](https://static.igem.wiki/teams/5425/drylab/fitting-parameters-rna-rna.webp)

Fig: The longer the antisense mRNA, the more spontaneous the Steric Blocking is. Of course, the simulation invokes approximations and assumptions, which are detailed in the Wet Lab Engineering Cycle.

2. The Spectrophotometric Assay showed a clear oscillation (note the kink!) of the growth curve in IPTG induction cases, but not in the control, quite in line with the expectation. This was a first remarkable observation for us. For then, the graph raised some questions. Firstly, the OD at which oscillation (or what we expect to be so) was observed, was at the same saturation OD as for the control (IPTG) case. But, in the IPTG cases, the growth was expected to oscillate about a lower saturation OD than in the control.

While analysing the result we obtained, we identified that one of key drawbacks of the experimental design is that, there MAY be manual error creeping in as for every reading, we take the culture off incubation, measure the OD, and keep a manual record of time all while. We switched to microplate reader for the first time.
The most probable take away from the Microplate Reader Assay is that, IPTG alone cannot prominently induce an oscillatory or stunted growth curve for the cells with our PCM construct. But, IPTG+dry tetracycline and IPTG+AHL could induce a fluctuation from the control curve. In fact, we could see a clear oscillation in the growth curve. This evidences our engineererd plasmid is working.
Analyzing the observation, we concluded that our tetR protein may be somewhat constitutively bound to the tetO region before the pTet promoter in the first transcription unit of our PCM construct and so only a very strong induction condition could cause the fluctuation in growth curve viz a viz the controls. A replicate experiment is underway, to confirm the results.
![](https://static.igem.wiki/teams/5425/experimental-assay/0-66f72baa51a1b.webp)

## Iteration 3: Circuit Designing: the Linalool Production Module (LPM)

As a part of our human practices, we had attended talks with several synthetic biologists across the globe. Before starting our wet lab work, we wanted solid verification from them. After several brainstorming sessions we came up with certain limitations of our design to produce Linalool. According to them, the MEV pathway can be successfully functioned if the chassis is pre-engineered to bear the stress of such a long enzymatic reactive pathway, alternatively we could order engineered or mutated strains to incorporate the MEV pathway. But due to time constraints and safety purposes, we discarded the solution of ordering a new strain for our experiments. Another path showed up when we were advised to manipulate the Methylerythritol pathway (MEP pathway). The MEP pathway is an innate metabolic pathway in E.coli that also produces isoprene for bacterial growth. Interestingly, the MEV and MEP pathway shares common intermediate substrates (DMAPP and IPP) among them, DMAPP being the former precursor of linalool. As it is clear that we could rather channelise the MEP pathway by just inserting GPPS and LIS into the bacteria to produce Linalool to reduce metabolic burden of the cloned organism.

## Iteration 4: Cloning and Expression

The HiFi Assembly Protocol was optimized for a successful engineering of plasmid PCM. The knowledge helped us calculate optimized HiFi assembly reaction mixtures for our future experiments.

## Iteration 5: Dry Lab: Initial Model for quorum sensing circuitI

We observed the oscillation in cell population in the presence of the quorum sensing circuit, thus verifying the presence of the negative feedback loop in it.
However, we also learned that our population equation could not relate death term, r0ct, with the accumulation of secondary metabolites and the reduction in substrate concentration as both r0 and c are constants and independent of the cell concentration as well as substrate concentration.
So, because this model cannot directly relate the accumulation of secondary metabolites and decrease in substrate concentration with the onset of the death phase in the population, we had to look upon developing an even better model.

## Iteration 6: Dry Lab: Final Model for quorum sensing circuit

We learned that our new growth model could very efficiently relate the death term of the population equation with the accumulation of toxic secondary metabolites and the reduction in substrate concentration.
We also observed that our quorum sensing model is able to give us the desired output of controlling the bacterial population at a threshold level quite below the carrying capacity of the original bacterial population without the quorum sensing circuit, and thereby, the population life/media potential of the culture is getting increased due to less accumulation of secondary metabolites and slower rate of substrate consumption, making the death phase of the population arrive at a much later stage than the original population.
Hence, our justification of choosing the later quorum sensing model to determine the bacterial population dynamics is accordingly verified.

## Iteration 7: Choosing Best Length of the Antisense

**Optimisation of PCR Protocol for Small Parts:**

The following modifications were incorporated:

1. The PCR reaction mixture was kept on ice. The PCR machine was allowed to preheat to 98°C, and then the PCR tubes were promptly put into the thermocycler. This reduced off-site binding.

2. Increasing Template (PCM extracted) concentration. That reduced primer dimers.

3. A control was kept with no template. The idea is that the primers, unreacted, would come down the gel electrophoresis and create primer dimers to help distinguish them in the other wells.

And excellent bright desired bands were observed in the gel.

The Microplate Reader Assay did show some excellent oscillatory growth curve upon induction, which was not there in the control case. For both 150 and 393 bp, the induction and fluctuations (oscillations) are visible. This indicates a success for our experiment.

We could not conclude if the assay of 150 or 393 bp showed a better oscillation, as both showed a similarly good result. We are yet to troubleshoot the 277 bp case.

![](https://static.igem.wiki/teams/5425/experimental-assay/antisense-exp-2-1-mm-iptg-66f72bb943dfe.webp)

◀

## Design

▶

## Iteration 1: Circuit Designing

Parallel to the linalool production module (LPM), we wanted an auto-regulated cell cycle regulation by another co-transformed plasmid. This plasmid will let each bacterium sense the cell density around itself. So that, when the bacterial density reaches an optimal threshold, a signal will trigger a feedback loop mechanism- which will cause cell cycle ‘arrest’. We needed two things.
Firstly, we needed a signalling molecule- which can be produced in proportion to the cell density outside- inside each bacterium itself. With the help of literature study, we came across the AHL molecule,
We identified the AHL to be utilised into the LuxI-LuxR Quorum Sensing Machinery of Vibrio fischeri1- which fulfilled our second need of the feedback loop, as well. All we needed to do was to transform this system into E. coli strain BL21.
![](https://static.igem.wiki/teams/5425/engineering/untitled-design-5.webp)

## Iteration 2: Cell Cycle Arrest (CCA)

Multiple strategies to trigger cell cycle arrest were discussed and tallied against our criteria of arrest- reversibility, speed, and non-toxicity to cells. A cell cycle death was hence ruled out, due to the rapid build up of toxic wastes that would make our (limited) food medium unusable.3 Hence, we went for a ‘softer’ strategy- simple, steric blocking of a key protein of cell cycle at its mRNA stage itself. Unlike DICER, etc. this strategy does NOT cleave the mRNA- it just prevents its translation. The following questions were raised:
Which gene to silence? We discussed an array of proteins involved in the replication fork of bacterial division4. Ultimately, we narrowed down to two- DnaB (a helicase) vs DnaA (initiator of cell replication). The knockout of the DnaB molecule was reportedly irreversible 5, pushing us to go for DnaA protein. Further, the DnaA molecule was BLASTed by our bio modellers and found to be a largely conserved protein- allowing us to silence the protein across a variety of strains of E. coli- including our expression strain BL21.
Which part of the DnaA’s mRNA should be sterically blocked? It was a million-dollar question. It was reported for a similarly long gene that about 400 bp long gene of the initial 5’ region (wrt coding strand), and also covering the Shine Dalgarno sequence upstream was sterically blocked and the gene’s expression could be silenced. 6 BLASTing, we could not find any information for the Shine Dalgarno sequence of this gene dnaA.
How long should the antisense mRNA be? Antisense Oligonuleotides were ruled on reported accounts of wide off site targeting despite their short size.6 A longer mRNA would mean more chances of effective blocking, but also more chances of secondary loop formation. We had to do our odds. The above paper6 motivated us into experimenting forward with the first 400 bp case.
We decided to silence the first 400 bp of the dnaA gene. (dnaA is the gene, DnaA is the protein). We designed a DNA sequence, under a pLux promoter, which coded for the antisense mRNA against the first 400 bp of the dnaA gene. It would sterically block its sense mRNA and hence stop dnaA at translation. Needless to say, no ribosome binding site was given. As we anyway don’t want its protein to be expressed.

## Iteration 3: Circuit Designing: the Linalool Production Module (LPM)

After thorough research for finding a biodegradable, eco-friendly, harmless overall green measure to manufacture our innovative cosmetic approach, we have found the most appropriate molecule fulfilling our desired properties in Linalool, a monoterpenoid mostly found in plants as a fragrant metabolite, moreover having antifungal and antimicrobial properties, a perfect combination for total foot care. Delving deep, we found that the plant enzymatic pathway known as Mevalonate Pathway (MEV Pathway) produces the Linalool where the direct enzyme Linalool synthase converts Geraniol diphosphate to Linalool. The MEV pathway is extensively researched and manipulated for different monoterpene production. We are grateful to the iGEM KEYSTONE China Team for their improved part for MEV pathway plasmid. Literature shows successful production of Linalool by incorporating the whole MEV pathways in bacteria. So we moved on building our model based on the design.

## Iteration 4: Cloning and Expression

We decided to go with the proprietary Hi Fi assembly offered by New England Biolabs. It is scarless, remarkably accurate and the we could leverage the experience of ex iGEMers from IISER-K and our advisors in this method. We identified that many of the Kit 2024 parts- had pSB13C backbone, with inserts at the same position. The backbone was chosen to be a pSB13C one, and the pair of linearization primers were cleverly designed so that they could linearise the backbones of most of those kit parts having pSB13C.

## Iteration 5: Dry Lab: Initial Model for quorum sensing circuitI

We wanted to determine the bacterial population dynamics using a set of deterministic ODEs. We chose all the necessary entities whose concentrations would be important to us, namely LuxI, LuxR, AHL, AiiA, A2R2 (AHL-LuxR complex), tetR, CCA (Antisense mRNA), N (Population concentration), S (Substrate concentration).

## Iteration 6: Dry Lab: Final Model for quorum sensing circuit

We proceeded to develop a population equation which could more efficiently relate the accumulation of secondary metabolites and decrease in substrate concentration with the onset of the death phase in the bacterial population. For that, we though of introducing an extra term to the logistic growth equation which would govern the onset of the death phase in the bacterial population.

## Iteration 7: Choosing Best Length of the Antisense

**Though we did incorporate a Cell Cycle Arrest (CCA) gene of length 400 bp length, we realized later that an empirical attempt at determining an even better Cell Cycle Arrest genes can be made.**

We stuck to DnaA reversible steric blocking, but now we decided to alter the length of the CCA gene, and hence the antisense mRNA. Ordering the replicates of the same PCM plasmid with different lengths of CCA was discarded in favour of a simple experiment. We wanted to put CCA genes of various lengths, such that they are expected to produce antisense mRNA against the first 150 bp, 278 bp, and 393 bp of the DnaA gene, starting from its AUG - each under an inducible promoter.

The part M20 of Kit Plate 1 of the 2024 Distribution kit had an IPTG inducible promoter pTac and LacO operator, we noticed after going through the Airtable. It also had a His operon terminator. So, we decided to leverage its backbone - we shall insert antisense of a certain length between this pTac + His operon terminator. We shall repeat this HiFi assembly for three such lengths of the CCA sequence.

![](https://static.igem.wiki/teams/5425/engineering/untitled-design-4.webp)

◀

## Build

▶

## Iteration 1: Circuit Designing

Once the circuit was built, the question of reversibility of Cell Cycle Arrest posed before us. Consider a situation wherein, the AHL concentration has been now enough to trigger cell cycle arrest, and so has happened. The signal is anyway getting produced, as per our circuit planned so far. But now, the signal is no more needed! How to decrease this signal?
Moreover, once the AHL signal is reduced, and as the cell density falls below the aforementioned threshold due to inevitable cell death, a reverse mechanism should restart Cell Cycle- which would then trigger another round of cell cycle arrest, and so on. We, thus, expect an oscillatory growth curve for the bacteria, oscillating around the logarithmic growth phase. How to have this reversibility? We used the tetR negative regulation of the Quorum Sensing.
We also decided to integrate a gene coding for the AiiA molecule, which is reported 2 to degrade the signal AHL. When cell cycle arrest occurs, tetR and AiiA both are produced. tetR represses pTetpromoter and hence downregulates AHL production, while AiiA degrades any AHL persistent in the environment at the moment.

## Iteration 2: Cell Cycle Arrest (CCA)

The 3rd fragment of our Population Control Module (PCM) circuit contained this Cell Cycle Arrest (CCA) sequence. It was ordered from our sponsor Twist BioScience and then, alongwith the other fragments, HiFi assembled into a pSB1C3 backbone. The assembled plasmids were then transformed into bacteria.

## Iteration 3: Circuit Designing: the Linalool Production Module (LPM)

We had put a little modification to the already built plasmid of the KEYSTONE Team according to our need i.e. to produce Linalool. We proposed to put GPPS and LIS gene by knocking out the idi gene from their design. The plan is to put DMAPP as a substrate of GPPS to produce the precursor of LIS (Linalool Synthase). For cloning the desired functional plasmid we ordered the pBbA5c-MevT (CO)-T1-MBIS(CO, ispA) . We have planned to knock out the ispA gene and insert GPPS and LIS in it. Hence we have ordered the mentioned plasmid from Addgene.
(Plasmid ordered from addgene: https://www.addgene.org/35152/sequences/)

## Iteration 4: Cloning and Expression

We decided to keep blunt overhangs in each fragment, so that they are Hi Fi compatible. The inserts of quorum sensing circuit were ordered in three parts from our sponsors- Twist Biosciences. The backbones were linearized out from Kit parts, as said.

## Iteration 5: Dry Lab: Initial Model for quorum sensing circuitI

We built the ODE system for the quorum sensing model to determine the bacterial population dynamics with time. The population equation in the presence of the quorum sensing circuit was formulated by modifying the logistic growth equation as such :-
\[ dN/dt = r0\*((1-μ[CCA])-ct/(1+(n\*(CAA))^n))N(1-N/K)\]
Here, r0 is the cell growth term and r0ct is the cell death term due to reduction in substrate concentration and accumulation of secondary metabolites, which eventually brings about the death phase in the population.
The \(r0\*(1-μ[CCA])\) term was incorporated to denote the reduction in the population growth rate due to the cell cycle arrest antisense mRNA (CCA).
The \(r0\*ct/(1+(n\*(CAA))^n)\) term was incorporated to denote the increase in the population life of the bacteria due to the population being controlled at a lower threshold.

## Iteration 6: Dry Lab: Final Model for quorum sensing circuit

We formulated the new population equation of the quorum sensing model, from the logistic growth model as :-
\[dN/dt= r0\*N/(1+(m\*[CAA])^r3)-r0\*(N^2)/K - aNt^4/((t^4+b^4)\*(1+(c\*S)^n))\]
Here, the first term is the growth term, being repressed proportionately by the concentration of the antisense mRNA (CCA). The second term is the natural death term, as usual.
The third term is the death term due to reduction in substrate concentration and the accumulation of secondary metabolites, which is responsible for bringing about the death phase in the bacterial population. It is multiplied to N as well as has a Hill term with respect to time to denote that the accumulation of secondary metabolites increases with increase with the cell population as peaks after a certain time. The term is also inversely proportional to the substrate concentration to indicate that the reduction in the amount of substrate amplifies the death phase of the population.

## Iteration 7: Choosing Best Length of the Antisense

**Build:** CCA of three different lengths - viz, 150 bp, 277 bp, and 393 bp - were amplified out, using primers, which were overlapping with the linearized M20 Backbone, from the main construct PCM (already having the maximum 400 bp arbitrary CCA sequence). And then one by one, they were HiFi assembled into three different plasmids. Let them be code named M20+CCA sequences of lengths 150 bp, 277 bp, and 393 bp, respectively.

During the amplification, the initial attempts at amplifying such small parts (100-400 bp) posed a challenge to us. We were initially unsuccessful in amplifying out CCA sequences of such short lengths. In the gel electrophoresis data, huge off-site binding and primer dimers were saddening us. By optimisation of protocol for PCR, we could successfully amplify them. Then on, the experiment proceeded smoothly.

◀

## Test

▶

## Iteration 1: Circuit Designing

Our math modellers ran an in-silico kinetic study on the above circuit.

## Iteration 2: Cell Cycle Arrest (CCA)

The testing of its efficacy was done through:
RNA-RNA Docking
Wet Lab: Once the Population Control Module (PCM) was constructed through Hifi Assembly, two assays were done to check the pattern of growth curve- whether an oscillatory growth is at all visible?
a spectrophotometric growth curve analysis, with IPTG induction. Three identical secondary cultures- one control, one with IPTG induction at 0th time point, and another with IPTG induction after 2 hours of setting the secondary culture, were given, for the E. coli strain BL21 cells transformed with PCM/Main Construct Hifi Assembled plasmid.
Microplate Reader Assay: A 96 well microplate was preplanned to contain transformed E. coli BL21 cells with the PCM, and then IPTG, IPTG+AHL, IPTG+Anhydrous Tetracycline in various concentrations in various wells, and in some wells, we had no inducer (as control).

## Iteration 3: Circuit Designing: the Linalool Production Module (LPM)

Due to time constraint, we could not perform the experiment regarding our newer approach to produce Linalool before our wiki submission, however, we have designed required primers in silico to linearize M20 plasmid from iGEM kit, amplify the gene insert GPPS and LIS and HiFi assembly the inserts with the plasmid backbone. The detailed primers and cloned in silico plasmids are documented in the Material part of our website.

## Iteration 4: Cloning and Expression

The HiFi Assembly was successfully done. Colony PCRs revealed the same. The plasmids were extracted and successfully transformed into DH5-alpha E. coli to increase copies. The plasmids extracted from their primary culture was then transformed into competent E. coli strain BL21 expression host. The construct has also been sent for a sequencing. We used this construct as the template for GFP characterization experiments and for the experiment to have an empirical idea on the best length of antisense mRNA. This is a successful engineering.

## Iteration 5: Dry Lab: Initial Model for quorum sensing circuitI

We ran a simulation of the model to determine the population dynamics of the bacteria with time. The N vs t plot is given below :-
![](https://static.igem.wiki/teams/5425/drylab/population-qs.webp)

## Iteration 6: Dry Lab: Final Model for quorum sensing circuit

We ran a simulation of the model to determine the population dynamics of the bacteria with time. The plots are given below:

![](https://static.igem.wiki/teams/5425/drylab/population-with-qs.webp)
![](https://static.igem.wiki/teams/5425/drylab/substrate-with-qs.webp)

The plots without the quorum sensing circuits are also given below:

![](https://static.igem.wiki/teams/5425/drylab/population-without-qs.webp)
![](https://static.igem.wiki/teams/5425/drylab/substrate-without-qs.webp)

## Iteration 7: Choosing Best Length of the Antisense

That the three assemblies were successful was confirmed through Colony PCRs and the plasmids were sent for sequencing. The plasmids were then transformed into DH5-alpha E. coli, extracted, and transformed into expression hosts E. coli strain BL21. Successful colonies were observed on chloramphenicol marker plates for 150 and 393 bp but NOT for 277 bp.

A similar microplate reader assay with induction by IPTG in various concentrations was done for 150 and 393 bp. And controls were given with no inducer.

![](https://static.igem.wiki/teams/5425/engineering/hifi-best-length-final.png)

Iteration 1

Iteration 2

Iteration 3

Iteration 4

Iteration 5

Iteration 6

Iteration 7

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![Inclusivity Banner](https://static.igem.wiki/teams/5425/page-elements/experiment-66faf2c4702ea.webp)

# Experiment

## Competent Cell Preparation

## Introduction

Preparing competent cells of *E. coli* strains BL21, DH5 alpha, and TOP10 involves a specific set of steps. Competent cells are vital for efficient transformation and DNA uptake during experiments.

## Materials

+ TFB-1 (pH 5.1) for 30 ml

| Component | Quantity |
| --- | --- |
| 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

| Component | Quantity |
| --- | --- |
| 10 mM MOPS | 0.031 g |
| 10 mM RbCl | 0.012 g |
| 15 mM CaCl2 | 0.124 g |
| 15% Glycerol | 2.25 ml |

## Procedure

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

### Caution

* All steps must be performed on ice.
* The lab hood should be bathed in UV light for more than 30 minutes before starting the procedure.

## Transformation

### Introduction

Transfer of miniprep plasmids or ligation mixture into *Escherichia coli* KRX or BL21.

### Materials

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

### Procedure

1. Take out the competent cells (100μl) 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.
3. Close the tube and mix them by gently tapping on the side of the tube.
4. Keep the tube in ice for 30 mins.
5. Set the hot water bath to 42°C and keep the SOC media in the incubator at 37°C.
6. Heat shock the tube for 45 seconds at 42°C, then place it back in ice for 5 mins.
7. Add SOC media to make up the volume to 1000μl and incubate it for 1.5 hours with shaking at 37°C.
8. Spread 100 μl on an agar plate and incubate at 37°C for 12-16 hours.

### Caution

The LB can be used as a media, but SOC is more favorable for bacterial growth and efficient colonies.

# Cloning Method

## For Plasmid Extraction: We followed the Qiagen Protocol.

## Notes before starting:

1. 1. Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
2. 2. Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
3. 3. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
4. 4. All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.

## Procedure:

1. 1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. 2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. 3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. 4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. 5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. 6.Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
7. **Recommended:** Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
8. **Note:** This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
9. 7. Transfer the QIAprep 2.0 spin column to the collection tube.
10. 8. Centrifuge for 1 min to rem/7ove residual wash buffer.
11. 9.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
12. 10. If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

To get a better yield, we strictly followed the following steps:

1. 1. After adding P2 buffer invert the tube vigorously and keep the tube still only for 1.5 min. (not more than that).
2. 2. Adding the N3 buffer again invert the tube 4-6 times and centrifuge it for 20 min at 13,000 rpm.
3. 3. Now carefully with the help of 20-200 uL pipette pull out the transparent part without disturbing the white part.
4. 4. Keep the spin column for 2 min in the rack and then spin it for 30 sec .
5. 5. Now have to add PB and keep the tube undisturbed for 5 min and again spin it for 2 min.
6. 6. Adding the PE buffer and spinning it three times.
7. 7. Now during the Elution we need to first add an elution buffer (20 ul) and keep the tube for 15 min and spin it for 2 min and we have to repeat the same process again.

## For Gel Extraction: We followed the Qiagen Protocol.

## Notes before starting:

1. 1. This protocol is for the purification of up to 10 μg DNA (70 bp to 10 kb).
2. 2. The yellow color of Buffer QG indicates a pH ≤+7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
3. 3. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
4. 4. Isopropanol (100%) and a heating block or water bath at 50°C are required.
5. 5. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

## Procedure

1. 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. 2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. 3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. 4. Add 1 gel volume isopropanol to the sample and mix.
5. 5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.
6. 6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
7. 7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
8. 8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. 9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let thecolumn stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
10. 10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

To get a better yield, we strictly followed the following steps:

1. 1. To get a better yield for the lengths of less than 600 bp we need to run the gel in presence of TBE buffer in 2.8 % gel media, which ultimately during extraction needs more than 5.5 % of QG buffer.
2. 2. Also need to use more than 1.5 % of the weight of isopropanol to minimize co precipitation of salt that interferes with downstream applications.
3. 3. Need to do centrifugation for 5 min at 13,000 rpm and instead of throwing the flow through spin it again for 1 min to extract the ultimate product from the sample.
4. 4. During the usage of the wash buffer spin it three times for 60 sec to get rid of all the ethanol from the solution.
5. 5. Do the elution with 55 degree centigrade hot NFW to reduce the amount of salt.

## Gel Electrophoresis

## Introduction

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.

## Materials

For Gel Electrophoresis

| Component |
| --- |
| 1x TAE Buffer |
| Agarose |
| EtBr (10mg/mL) |
| Gel Cast |
| Gel Combs |

## Procedure

1. To make 1% Agarose gel, mix 1g of Agarose in 100ml of 1X TAE.
2. To dissolve, microwave it for 2-3 mins until it completely dissolves with occasional swirling. Remember not to over-boil as some amount can get evaporated and spilled.
3. Let it cool to 50°C.
4. Add EtBr to a final concentration of 0.2 - 0.5 μg/ml.
5. Cast the gel in the gel tray and place the comb carefully.
6. Keep the gel at room temperature for 20-25 mins (till it solidifies properly).
7. Add loading dye to each sample.
8. Fill the gel box with 1X TAE buffer, then place the solidified agarose gel in the gel box . Remember to check if the gel is fully covered with the buffer, if not so then add some more buffer!
9. Load the ladder in the first lane. Then carefully load samples.
10. Run the gel at 80-120 Volt until the dye line is approximately 75-80% of the way down the gel. Should take 1-1.5 hrs approximately.
11. Remove the Gel carefully and then visualize it using UV.
12. Now add 1 ml of autoclave water to the 1 mg of the etbr and vortex it until the complete ppt has gone .

### Caution

* For EtBr Prep - Wear goggles and maintain a safe distance.

## Table: Agarose Gel % and Length of DNA to resolve.

| Agarose Gel % | Length of DNA to resolve |
| --- | --- |
| 0.5 | 1000 - 30000 bp |
| 0.7 | 800 - 12000 bp |
| 1.0 | 500 - 10000 bp |
| 1.2 | 400 - 7000 bp |
| 1.5 | 200 - 3000 bp |
|
 2.0 | 50 - 2000 bp |

## Polymerase Chain Reaction (PCR)

## Amplification PCR

## Introduction

PCR is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing us to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail.

## Materials

For Amplification PCR

| Component |
| --- |
| Autoclaved PCR tubes |
| Agar Plates (with desried antibiotics) |
| NEB Q5® High-Fidelity 2X Master Mix |
| Micropipette tips |
| Respective Primers |
|
 NFW |

## Procedure

1. 1. Prepare the PCR mix by adding the following reagents by placing the PCR tubes all on ice.

| Reagents | Volume (μL) |
| --- | --- |
| NEB Q5® High-Fidelity 2X Master Mix | 25 |
| Forward Primer | 2.5 |
| Reverse Primer | 2.5 |
| Template DNA | 3 |
| NFW | 17 |

- 2. Rest of the protocol to be followed as provided by NEB (linked in references).

## Gradient PCR

## We have followed the same protocol as the NEB provided (linked in references).

## Colony PCR

## Introduction

Colony PCR is a method for rapidly screening colonies of bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.

## Materials

For Colony PCR

| Component |
| --- |
| Autoclaved PCR tubes |
| Agar Plates (with desried antibiotics) |
| Go Taq Green PCR master-mix by Promega |
| Micropipette tips |
| VF2 and VR primer. |
|
 NFW |

## Procedure

1. 1. Fill PCR tubes (as many required) with 10μl of NFW in each tube.
2. 2. The culture plates with colonies were marked accordingly.
3. 3. One colony was picked with a sterile tip and was streaked at the marked position on the new plate.
4. 4. Then put the tip in the PCR tube containing 10μl of NFW.
5. 5. Take the same number of autoclaved PCR tubes as number of colonies picked and label accordingly.
6. 6. Add the following reagents in the PCR tubes to make a reaction mixture of 25μl.

| Reagents | Volume (μL) |
| --- | --- |
| Go Taq Green DNA master mix | 12.5 |
| VF2 | 1 |
| VR | 1 |
| Template DNA | 5 |
| NFW | 5.5 |

- The only difference is that we have to set the denaturation temp at around 95 degrees centigrade for 10 min.

## PCR Optimised for Amplification of Small Lengths of DNA

## Introduction

In the very beginning of the reaction, we have to heat the lid and PCR machine up to 98 degrees Celsius and then from the ice directly have to put the tubes inside the PCR machine.

Then follow the regular rule of PCR.

This is basically optimized PCR protocol for amplification of the short lengths (100-400 bp).

## HiFi Assembly

## Introduction

NEBilder HiFi Assembly: To assemble the inserts and the backbone and check the induction.

## Optimal Quantities

NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool, NEBiocalculator (linked in references).

pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)

50 ng of 5000 bp dsDNA is about 0.015 pmols

50 ng of 500 bp dsDNA is about 0.15 pmols

The mass of each fragment can be measured using the Nanodrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.

## HiFi DNA Assembly Protocol

1. 1. Set up the following reaction on ice as follows.

Headings: Recommended Amount of Fragments Used for Assembly

|  | 2–3 Fragment Assembly\* | 4-6 Fragment Assembly\*\* | NEBuilder Positive Control✝ |
| --- | --- | --- | --- |
| Recommended DNA Molar Ratio | vector: insert = 1:2 | vector: insert = 1:1 |  |
| Total Amount of Fragments | 0.03–0.2 pmols\* X μl | 0.2–0.5 pmols\*\* X μl | 10 μl |
| NEBuilder HiFi DNA Assembly Master Mix | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl | 20 μl | 20 μl |

2. 2. Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.
3. To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with equimolar of all fragments (suggested: 0.05 pmol each).
4. Control reagents are provided for 5 experiments.
5. If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NE Builder HiFi DNA Assembly Master Mix.

## Single Digestion

## We recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl. A typical restriction enzyme digestion protocol is below.

## Protocol:

1. Add the following reaction components in the order indicated:

| Component | Volume (μL) |
| --- | --- |
| Water, nuclease-free | 16-16.5 |
| 10X recommended buffer for restriction enzyme | 2 |
| Substrate DNA | 1 (~1 µg) |
| Restriction enzyme | 0.5-1 (5-10 u) |
| Total | 20 |

- 2. Mix gently and spin down briefly.
- 3. Incubate at the optimal reaction temperature for 1-16 hours.
- Note: The digestion reaction may be scaled either up or down. Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

## References

1. <https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/8-DNA-ladder-tips.html>
2. <https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492>
3. <https://ww.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol>
4. [NEBiocalculato](https://www.neb.com/en/external-links/nebiocalculator)
5. <https://www.neb.com/en/protocols/2018/07/30/restriction-digest-protocol>
6. <https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Design{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

# Results

[Overview](#section0)
[Construction Of PCM](#section3)
[Characterisation of PCM Circuit](#section4)
[Antisense](#section5)
[Quantitative Assay](#section1)
[Fungal Assay](#section2)

## Overview

> “If I had thought about it, I wouldn’t have done the experiment. The literature was full of examples that said you can’t do this.”
>
> - Spencer Silver

Synthetic Biology experimentation remains the core root of iGEM. To realise our hypothesised product, the team IISER Kolkata has done extensive wet lab work in their dedicated laboratory over the last few months. The results of the same are attached herewith. After the brainstorming sessions and finalization of the project, learning up new lab techniques was the big fun challenge for the wet lab members. In the short period, the lab produced its first construct of plasmid- the Population Control Module. Following which, many more experiments, plasmid constructions and characterisations were held. They were complemented with assays using the latest equipment available in our laboratory. We leveraged the common facility of our Department of Biological Sciences as and when needed.

## Construction of PCM(Population control module)

## **Cloning of pSB1C3**

**Aim:** To procure higher quantities of pSB1C3 backbone that will be subsequently used for the NEBuilder® HiFi DNA assembly.

**Experiment:** We have used self-chemically prepared DH5-alpha chemically procured competent cells for cloning the revived part C-11 (kit plate 1, DistributionKit 2024) pSB1C3 via chemical transformation, did a plasmid extraction to get a better yield.

**Result:** We have successfully cloned the pSB1C3 vector through transformation and then done a plasmid extraction.

**The concentration of the vector:** 256.6 ng/ul (Nanodrop concentration)

## **PCR Amplification of the Gene Fragments**

**Aim:** To PCR amplify the three fragments (from TwistBioScience) (code named by us as insert-1, insert-2, insert-3) so then we can perform the NEBuilder® HiFi DNA assembly of them with the pSB1C3 vector.

**Experiments:**

1. We did a gradient PCR to determine the annealing temperature for each fragment with the respective forward and reverse primers.

2. We proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them.

3. For insert-2, we observed non-specific binding, which we managed to remove by running the gel 30 minutes more.

**Result:** 62.5 degrees Celsius was the optimal temperature for insert-1, insert-2, and insert-3.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/insert-1-and-2-grad-66f72dcf1fcf0.webp)
****Fig****- the Gradient of the insert-1 and insert-2

| Insert | Sequence | Base Pairs (bp) |
| --- | --- | --- |
| Insert-1 | Ptet-RBS-LuxI-terminator-ptac-RBS-LuxR-terminator-pLux-RBS | 1711 bp |
| Insert-2 | tetR-RBS-AiiA dtag | 972 bp |
| Insert-3 | AiiA-dtag-terminator-pLUX-RBS-Antisense-terminator | 1164 bp |

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/insert-3-gradient-66f72dd05eec5.webp)
**Fig**-The insert – 3 gradient PCR gel run and the desired bright band is on the 62 degree Celsius

****Fig**:** The insert-3 gradient PCR gel run and the desired bright band is on the 62-degree Celsius.

For gel extraction, we had to run the gel for more than 1 hour to avoid non-specific binding. Using DMSO for the gel run at a lower voltage for 1 hour is much more favorable (optimized).

**The gel extracts had the following purities:**

| Insert | Purity (ng/ul) |
| --- | --- |
| Insert-1 | 41.9 ng/ul |
| Insert-2 | 126 ng/ul |
| Insert-3 | 106 ng/ul |

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/insert-3-gel-run-66f72dcf19dfd.webp)
**Fig**- the amplified insert -3 gel run
![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/insert-1-and-2-66f72dd080448.webp)
**Fig**- insert 1 and insert 2 circuit PCR amplified gel run

**Linearisation of the plasmid**

**Aim** - to linearize the plasmid through PCR reaction for the NEB HiFi assembly.

**Experiment** - we did a gradient PCR to determine the annealing temperature for pSB1C3 with the respective forward and reverse primers. Then we have proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them. The optimal annealing temperature was found to be 60 degree Celsius.

**Result** - we have achieved a desired length in the gel imaging and through the HiFi assembly of this vector lead to a successful circuit assembly.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/linearisation-gel-66f72dd15ce09.webp)
**Fig**- the gel extraction of the PCR amplified plasmid
Concentration of extracted plasmid= 56ng/u
![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/linearisation-66f72dd1db378.webp)
**Fig**-gradient PCR of the plasmid backbone

**NEBuilder HiFi Assembly**

**Aim** – to assemble the inserts and the backbone and check the induction.

**Experiments** -

![](https://static.igem.wiki/teams/5425/experimental-assay/screenshot-2024-09-29-115417.webp)

**Source**: NEB Website

Incubate samples in a thermocycler at 50°C for 60 minutes. Following incubation, store samples on ice or at –20°C for subsequent transformation.

**Result** – Single Digestion of the HiFi assembly circuit in the BamHI circuit and PCR run of the circuit show the desired bands on insert-1, insert-2, and insert-3.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/single-digestion-66f72dd2dc5af.webp)
**Fig**- Digestion of the plasmid (assembled) and the PCR run of the PCM (Main Construct) construct

To check the induction of the circuit we have performed an IPTG induction experiment which is further analysed in the quantitative assay.

## Snap Gene file for the Entire Assembly – length 5908 bp

![](https://static.igem.wiki/teams/5425/screenshot-2024-10-02-011843.png)
**Fig**- The Whole HiFi Assembled Circuit

**By optimizing the previously mentioned protocol, we achieved successful cloning, which was further verified through single digestion at BamHI and colony PCR. These validation steps confirmed that the cloning was successful.**

## Characterisation of PCM Circuit

## Cloning of the Circuit

**Extraction of the GFP Plasmid**

**Aim** – To procure higher quantities of sfGFP (superfolded GFP) that will be subsequently used for the NEBuilder® HiFi DNA assembly.

**Experiment** – We have used self-prepared DH5 alpha chemically procured competent cells for cloning the sfGFP via chemical transformation. Moreover, we did a plasmid extraction to get a better yield.

**Result** – We have successfully cloned the sfGFP sequence through transformation, followed by plasmid extraction. The concentration of the plasmid extracted sfGFP was 429 ng/ul.

## Amplification of the GFP

**Aim** – To amplify the sfGFP sequence through the Polymerase Chain Reaction (PCR) for a future HiFi assembly.

**Experiment** – We have done a gradient PCR to determine the annealing temperature for sfGFP with the respective forward and reverse primers. We then proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them. The optimal annealing temperature was 57 degree Celsius. The gradient PCRs for the sfGFP characterization of the tetR-AiiA sequence and sequence for Antisense DNA have been carried out with the following temperatures:– 52, 54, 56, 58, 60 degree Celsius.

**Results** – The desired band is observed at a length of around 757 bp. To get a better result in the gel electrophoresis experiment, we have used TAE buffer, which is not practically suitable for a long run. The gel was then run by reducing the voltage and increasing the time.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/copy-of-gradient-66f72dd7554c5.webp)

## Linearisation for addition of GFP to the downstream of pLUX of AiiA for its characterisation (tetR)

**Aim** - To linearise the tetR-AiiA sequence characterisation part through PCR so as to obtain the backbone for insertion of sfGFP through NEBuilder® HiFi DNA assembly reaction.

**Experiment** - We have done a gradient PCR to determine the annealing temperature with the respective forward and reverse primers. We have then proceeded with the optimal annealing temperature of the respective fragments to PCR amplify them. The optimal annealing temperature is 57 degree Celsius. The gradient temperatures are set at: 50, 53, 56, 58, 60, 62 degree Celsius.

**Result** - We have achieved the desired length on gel imaging. We have obtained a successful circuit assembly through HiFi assembly of this vector.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/tetr-gfp-66f72dd5396d6.webp)

## Linearisation for addition of GFP in the place of Antisense for its characterisation (Antisense)

**Aim** - To linearise the Antisense characterisation part through PCR to insert sfGFP in the main circuit through NEBuilder® HiFi DNA assembly reaction.

**Experiment** - We proceeded with a gradient PCR to determine the annealing temperature for the linearised plasmid (up to the sequence of Antisense DNA) with the respective forward and reverse primers. We have then proceeded with the optimal annealing temperature of the respective fragments to amplify them with PCR reaction. The set gradient temperatures are 50, 53, 58, 60, and 62 degrees Celsius. The optimal annealing temperature is 58 degrees Celsius.

**Result** - We have achieved the desired length on gel imaging. We have obtained a successful circuit assembly through HiFi assembly of this vector.

## Amplification of GFP in the pLUX of AiiA for its characterisation (tetR) and GFP in the place of Antisense sequence for its characterisation (Antisense)

**Aim** - To amplify the Antisense and tetR sfGFP characterisation part through PCR to get a better yield in the HiFi.

**Experiment** - We have proceeded with the optimal annealing temperature of the respective fragments (inferred from previous gradient PCRs) to PCR amplify them and then performed gel extraction in 0.8-1.5% agarose gel media. For gel extraction of length around 4-5 kb, we ran the gel on the TBE buffer and extracted the gel with 1.5% weight of isopropanol.

**Result** - We have observed that the gel bands have come on particular desired lengths. Being cautious while performing gel extraction brings us a step closer to a successful HiFi assembly.

| Insert | Purity (ng/ul) |
| --- | --- |
| Gfp-tetR | 38.8 |
| Amp-tetR | 35.4 |
| GFP-anti | 85.9 |
| Amp-anti | 32.8 |

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/copy-of-gel-extr-66f72dd8307ff.webp)

## HiFi assembly of the whole circuit

**Aim** - To assemble and characterize the full circuit besides checking whether it produces our protein of interest or not.

**Experiment** -

For Antisense sequence set up reaction on ice:

| Component | Volume | Amount |
| --- | --- | --- |
| Anti-amp (vector) | 6.7 ul | 0.067 pmoles |
| Sf-GFP | 0.7 ul | 0.133 pmoles |
| Deionised water | 2.6 ul |  |
| NEBuilder HiFi DNA assembly Master Mix | 10.0 ul |  |

**Total volume** - 20 ul

For the tetR set up reaction on ice:

| Component | Volume | Amount |
| --- | --- | --- |
| tetR-amp (vector) | 4.3 ul | 0.067 pmoles |
| Sf-GFP | 1.6 ul | 0.133 pmoles |
| Deionised water | 4.1 ul |  |
| NEBuilder HiFi DNA assembly Master Mix | 10.0 ul |  |

**Total volume** - 20 ul

The reaction mixtures will be incubated around 50 degrees Celsius for 15 minutes in the PCR machine.

**Result** - Colony PCR and quantitative assay analysis give positive results on production of the protein of interest.

**The snap gene files of the above characterisation –**

| Name |  | Snap Gene file Length |
| --- | --- | --- |
| GFP after pLUX of AiiA to characterise the circuit |  | 4475 bp |
| GFP instead of antisense to characterise the circuit |  | 6097 bp |
| sfGFP |  | 717 bp |
| Amplified GFP |  | 757 bp |

## Antisense

## Experiments held to determine the best possible length of the cell cycle arrest (CCA) sequence (aka the Antisense)

| Length of CCA Gene | Optimal Annealing Temperature (°C) |
| --- | --- |
| 150 bp | 60 |
| 277 bp | 60 |
| 393 bp | 66 |

**Aim** - to amplify the CCA gene sequence of different lengths from the 400 bp CCA as a part of the PCM construct through the PCR for the NEBuilder® HiFi DNA assembly into M20 pSB1C3SA backbone.

**Experiment** - A Gradient PCR (on a common gradient of annealing temperatures, across 54, 56, 58, 60, 62 deg C) was done to determine the annealing temperature for CCA gene for different length with the respective forward (FP common) and reverse primers (variable). Then we have proceeded with the optimal annealing temperatures of the respective fragments to PCR amplify them.

**Result:** The optimal annealing temperature is found to be 60, 60, 66 deg C for the 150 bp, 277 bp, 393 bp, respectively.

![](https://static.igem.wiki/teams/5425/wet-lab/150-bp-gpcr.webp)
**Fig**: Gradient PCR of CCA of length 150 bp. Similarly done for the other two cases. (Ladder= NEB 1 kb+)

## Amplification Of The Respective Different Lengths Of Antisenses/CCA

**Aim** - to amplify the Antisense three different lengths part through PCR to get a better yield in the HiFi result during NEBuilder® HiFi DNA assembly reaction.

**Experiment** - The amp. PCR protocol was troubleshot for small parts. The reaction was made to simulate ‘Hot Start’ – i.e., the reaction mixture was kept on ice. The PCR machine was preheated to 98 deg C and then paused. The reaction (100 µL, divided into two 50 µL tubes for a better reaction) tubes were then suddenly put into the PCR machine.

**Result** - The three lengths of the CCA were respectively run on suitable gels, and excellent bands at desired lengths were obtained, which were excised and immediately extracted with excellent yield (concentration) of DNA.

![](https://static.igem.wiki/teams/5425/wet-lab/150-bp.jpg)
**Fig**- Amplification PCR Pdt. of CCA of 150 bp (Ladder= NEB 1 kb+)
![](https://static.igem.wiki/teams/5425/wet-lab/393-bp.webp)
**Fig**: Amplification PCR Pdt. of CCA of 393 bp (Ladder= NEB 1 kb+)
![]( https://static.igem.wiki/teams/5425/wet-lab/277-bp.webp)
**Fig**: Amp. PCR Pdt. of CCA of length 277 bp (Ladder= NEB 1 kb+)

## Linearization of Vector for CCA of Variable Lengths

**Aim**: To run gradient PCR for the linearization of the common backbone vector for each of our antisenses (Kit part M20, Kit Plate 1, Kit 2024).

**Theory**: The primers were based on the amplification of a major part, including the pTac promoter, lacO operator, and a terminator. The pTac was default suppressed due to lacI in the environment but can be switched on by IPTG. The idea was to exploit this kit part into our attempt at switching OFF - ON - OFF systems of our antisenses when we would assemble our antisenses (of various lengths) into this backbone.

**Result**: The best Ta for the M20 Backbone was found to be 63 deg C.

**Aim**: To linearize the M20 Backbone based on the Ta obtained earlier.

**Experiment**: A 100 µL reaction mixture, as written earlier, was set up. It was divided into two 50 µL reaction tubes for better PCR (a recent learning) and then loaded onto a single, big well for a total of 100 µL.

**Result**: Visualized on a low percent gel.

![](https://static.igem.wiki/teams/5425/experimental-assay/gel-pics/linearisation-m20-wbep.jpg)
**Fig**: Linearization PCR Pdt. Of M20 Backbone (Ladder= NEB 1 kb+)

## NEB HiFi Assemblies

### HiFi 1: (M20+150bp)

**Vector Length**: 2,311 bp

**Vector Source**: M20, Kit Plate 1, Distribution Kit 2024

| Insert | Length (bp) | Nanodrop Conc. Of Gel Extract (ng/uL) | HiFi Assembly used in |
| --- | --- | --- | --- |
| CCA of 150 bp | 150+~40 overhang | 62.25 (dil. 2X) | HiFi 1 |
| CCA of 277 bp | 277+~40 overhang | 17.9 | HiFi 2 |
| CCA of 393 bp | 393+~40 overhang | 79.8 | HiFi 3 |

The following reaction mixtures were set up:

#### HiFi 1

* **M20 Vector**: 1 µL
* **Insert 150 bp**: 0.3 µL
* **NEB HiFi Builder MasterMix**: 6 µL
* **Deionized H2O**: 12.7 µL
* **Total**: 20 µL

#### HiFi 2

* **M20 Vector**: 1.26 µL (0.05 pmolar)
* **Insert 277 bp**: 0.6 µL (1 pmolar)
* **NEB HiFi Builder MasterMix**: 6 µL
* **Deionized H2O**: 12.2 µL
* **Total**: 20 µL

#### HiFi 3

* **M20 Vector**: 2.8 µL
* **Insert 393 bp**: 0.24 µL
* **NEB HiFi Builder MasterMix**: 6 µL
* **Deionized H2O**: 12.6 µL
* **Total**: 20 µL

The backbone, the fragment (either of antisense 150 bp, 277 bp, 393 bp), MasterMix, and NFW were incubated in the PCR machine at 50 deg C for 30 min.

## Quantitative Assay

Spectrophotometric Analysis

Aim – to observe induction of the IPTG inducible promoter pTac and its transcription unit in the PCM (Main Circuit) through a growth curve measurement of the PCM-transformed bacterial culture.

Experiment – a spectrophotometric growth curve analysis, with IPTG induction. Three identical secondary cultures- one control, one with IPTG induction at 0th time point, and another with IPTG induction after 2 hours of setting the secondary culture, were given, for the E. coli strain BL21 cells transformed with PCM/Main Construct Hifi Assembled plasmid.

Result- there is a minute deserved oscillation in the circuit is observed.

![](https://static.igem.wiki/teams/5425/experimental-assay/od-66f72bc3d33a7.webp)

Microplate Assays

Aim- to observe how different concentration of inducer IPTG affected the growth and production of the CCA (Cell Cycle Arrest) sequence (aka the Antisense)

Exp.- in a 24 well microplate with a different concentrations of IPTG – 0.1 mM , 0.5 mM and 5 mM- we have done the assay for the both control (revived part M20, kit plate 1, 2024 Kit ) and PCM construct (aka the Main Construct).

Result- the induction in the circuit is not that prominent but a minute decline in the stationary phase population was observed wrt control.

![](https://static.igem.wiki/teams/5425/registry/5-mm-iptg.webp)

Experiment-2

Aim- to observe how different concentration of inducer IPTG , AHL and aTc (anhydrous Tetracycline) affected the growth and production of antisense (CCA)

Experiment- in a 96 well microplate with a different concentration of IPTG – 0.1 mM , 0.5 mM and 5 mM we have done the assay for the both control M20 ( well plate 1 ) and PCM construct now we have given diff concentration of AHL and same concentration- aTc (anhydrous tetracycline)

Result-
![](https://static.igem.wiki/teams/5425/description/1-mm-iptg-ahl-and-atc.jpg)
**Fig** - the aTc induced circuit shows an observable fluctuation. (HiFi denotes the Main Construct or the PCM)
![](https://static.igem.wiki/teams/5425/0.jpg)
**Fig**- the without atc induced circuit show an observable fluctuation.

Experiment- 3 Fluorescence Assay-

Aim- characterisation of the circuit through fluorophore sfGFP through the fluorescence analysis

Experiment- in a well microplate with a different concentration of IPTG – 0.1 mM , 0.5 mM and 5 mM we have done the assay for the both control M20 ( well plate 1 ) and PCM construct.

We replaced tetR\_AiiA-dtag under pTac promoter, in our construct- the PCM, with sfGFP, and code-named the construct as tetR. It showed the following growth curve with IPTG induction for the 1 mM case.
![](https://static.igem.wiki/teams/5425/description/tetr-at-1-mm-iptg.jpg)
**Fig**- the induced tetR or the circuit is successfully characterised in presence of IPTG
![](https://static.igem.wiki/teams/5425/description/tetr-at-0.jpg)
**Fig**- the induced tetR is successfully characterised in presence of IPTG.
![](https://static.igem.wiki/teams/5425/description/antisense-1-mm.jpg)
**Fig**- the induced ‘anti’ circuit (sfGFP replacing CCA sequence downstream of second pLux promoter) is successfully characterised in presence of IPTG.

![](https://static.igem.wiki/teams/5425/experimental-assay/data-5.webp)
![](https://static.igem.wiki/teams/5425/experimental-assay/anti-at-200-ng-ml-atc-conc-66f72bb088cde.webp)
**Fig**- fluro/OD vs time graph where with the increase of the concentration of the atc a major difference has been observed.

Experiment- 4

Oscillatory growth curve was achieved in different length analyses, quite in line with our expectations.

![](https://static.igem.wiki/teams/5425/antisense-exp-1-0.jpg)
![](https://static.igem.wiki/teams/5425/antisense-exp-2-1-mm-iptg.jpg)
![](https://static.igem.wiki/teams/5425/antisense-exp-3-2-mm-iptg.jpg)
![](https://static.igem.wiki/teams/5425/antisense-exp-4-5.jpg)
Analysis of the Resultant data
We eventually fitted our wetlab data obtained from 0.1nM IPTG+AHL induction with the math model for quorum sensing and came up with the following fitted plot. For an insight into the fitted parameters, visit DryLab

![](https://static.igem.wiki/teams/5425/drylab/fitted-control-fitted-qs-2.webp)
We did not observe significant changes in fluorescence during our assay, indicating that the circuit did not function as expected and the components were not properly fine-tuned. This opens the opportunity for circuit modifications, where we plan to introduce degradation tags to various components to better fine-tune parameters based on a revised model. Initial predictions, based on our circuit's original parameters, forecasted oscillations, but experiments revealed no oscillations or notable phenotypic changes. This failure could be due to signals being too weak for detection by our instruments. However, we did demonstrate that induction of the cca gene caused an arrest, which was released after a specific time interval. The mechanism behind this release remains unclear, but we have designed experiments to explore it further.

## Fungal Assay

Aim- to check the growth of the fungi (Chaetomium thermophilum ) with respect to different concentration of the commercial linalool (monoterpenoid).

Experiment-to check the growth we need to prepare a suitable media for fungus

Mycelium basically propagates on the CCM medium.To prepare this we need to follow the following composition:

| Components | Amount |
| --- | --- |
| Sucrose | 3 g |
| NaCl | 0.5 g |
| K₂HPO₄ · 3H₂O | 0.65 g |
| Magnesium sulfate heptahydrate | 0.5 g |
| Fe(III) sulfate hydrate | 0.01 g |
| Tryptone | 5 g |
| Peptone | 1 g |
| Yeast extract | 1 g |
| Dextrin | 15 g |
| Agar | 20 g |
| Distilled water | 1 Litre |

pH must be 6.5-7.5

## **Procedure:**

1. We need to grow the fungus overnight in a culture and through the pipette pluck one single colony in the centre of the plate.

2. After that, marking the same distance with the scale, we need to put the single colony in the plates varying the concentration of the Linalool on the plates.

3. The plates need to be incubated under 45 degrees centigrade at 250 rpm.

4. After 24 hrs, we have to check the growth and measure the lengths of the radius to get a rough idea about the growth of mycelium.

## **Result:**

With the varying concentration of Linalool, we have found that at a certain level of concentration, fungal growth has been stopped. The below observations are after 24 hrs.

![](https://static.igem.wiki/teams/5425/wet-lab/1.png)
**Fig**- after treatment with 0 linalool mycelia growth diameter is 55 mm
![](https://static.igem.wiki/teams/5425/wet-lab/2.png)
**Fig**- after treatment with 56 ug/mL the mycelia growth diameter is 30 mm
![](https://static.igem.wiki/teams/5425/wet-lab/3.png)
**Fig**- after treatment with 150 ug/mL Linalool the mycelia growth diameter is 25 mm
![](https://static.igem.wiki/teams/5425/wet-lab/4.png)
**Fig**-After treatment with 256 ug/ml Linalool no growth observed

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