iGEM 2023 — Team svce-chennai

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## NOTEBOOK

January

February

March

April

May

June

July

August

September

October

### January

January was an exciting month for our team as we embarked on the journey of selecting our team members and finalizing our team composition.

* January 9, 2023: Our Mentors explained about iGEM and the previous achievements of our teams in the iGEM Competitions
* January 10, 2023, to January 15, 2023: We surged through our previous team's Wiki pages and a few other teams in order to get an idea of how an idea about the competition and how an idea has to be developed
* January 16, 2023, to January 27, 2023: The team selection process started
* January 28, 2023: We selected our team members and finalized our team
* January 30, 2023: We got the list of chemicals in the department and made a note of the chemicals that we can use for the preliminary experiments.

### February

* February 1, 2023: We started working on our project with full force.
* February 6, 2023, to February 15, 2023: Brainstorming sessions
* February 17, 2023, to February 22, 2023: Idea formulation
* February 23, 2023, to February 25, 2023: We framed a preliminary budget with the list of chemicals that we will be needing for our experiments.
* February 27, 2023 to March 6, 2023: CAT 1 Examination- No work was carried out.

### March

* March 13, 2023, to March 24, 2023: we had a collection of promising project ideas that held the potential to make a difference in various fields
* March 27, 2023, to March 31, 2023: selection of organisms, pathways, and genome sequences that would serve as the basis for our project
* March 23, 2023, to March 29, 2023: We learned about tools like SnapGene, which was valuable in our project design phase.

### April

* April 13, 2023, to April 19, 2023: CAT 2 Examination- No work was carried out.
* April 20, 2023, to April 21, 2023: We presented our initial ideas to all colleagues, regardless of their year, and to all the faculties of our department
* April 24, 2023, to April 28, 2023: After carefully considering the inputs from our faculties, we finalized our idea. We took into account feasibility, novelty, and potential impact when making our final decision.

### May

May was a month filled with exciting progress for our iGEM team!

* May 3, 2023: In order to secure sponsors for our project, we conducted an extensive search to identify potential partners who shared our vision and goals.
* May 14, 2023: 1st iGEM alumni meet with Prasanna- gave us some input on funding, outreach, and human practices.
* May 15, 2023: 1st official meeting to divide subteams and select leads along with our instructors and mentors
* May 17, 2023: Insta Page Launch, We encourage you to follow us on Instagram [@igem\_svce\_chennai] to stay updated on our journey and be a part of our iGEM project.
* May 18, 2023, to May 23, 2023: CAT 3 Examination- No major work was carried out.
* May 21, 2023: iGEM alumni meet with Karthick- for input on ideas, biosafety, what to do and what not to do, ideas for ighp, and a few more contacts to approach regarding gene construct.
* May 24, 2023: iGEM alumni meet with Veveeyan- complete lab instructions on how to build the gene circuit. We figured out a few problems with the idea, and then we did some literature surveys to refine the idea.

### June

* June 5, 2023, to June 19, 2023: End Semester Examinations
* June 13, 2023: To gather additional resources for our project, we organized a crowdfunding campaign through an online platform called Milaap.
* June 29, 2023: Meet with Alumni Rajeshwar Nitiyanandan- We presented our idea and sponsorship opportunity.
* June 20, 2023: We actively reached out to potential sponsors who were interested in supporting our iGEM team and our project.
* June 21, 2023: We submitted our initial project safety form
* June 24, 2023: We conducted surveys to gain insights from our target audience, and the feedback we received allowed us to make informed decisions as we moved forward with our research and implementation plans.

### July

* July 5, 2023: Our team has been shortlisted for the Team Impact Grant 2023!
* July 1, 2023, to July 7, 2023: Plastic-Free Month Instagram Quiz
* July 28, 2023: World Nature Conservation Day Collaboration
* July 30, 2023: Plastic-Free July Beach Cleanup
* July 28, 2023, to July 30, 2023: Our team participated in the All India iGEM Meet at IISER Bhopal
* July 24, 2023, to August 10, 2023: Schools and Colleges visits

Throughout July, we've been on a mission to make a positive impact on our planet. #GreenJulyJourney. Visit our Human Practices page to learn more about the above-mentioned events.

### August

* August 21, 2023 to August 23, 2023: CAT 1 Examinations- No work was carried out
* August 24 and 25, 2023: Our Bioinformatics and Synthetic Biology workshop- BIOBLITZ- Phase I
* August 30, 2023: Our team members had a chance to present our idea at the FICCI-Summit & Awards
* August 31, 2023: We received the ordered chemicals to carry out the experiments

### September

* September 1, 2023: Primers and kits have arrived.
* September 8, 2023, to September 12, 2023: Promotional video - Ideation, Filming & Uploading
* September 13 & 14, 2023: BIOBLITZ-Phase II
* September 17, 2023: Human Practices event- “Biopark”
* September 20, 2023: Started filling out all the forms (Deliverables)
* September 24, 2023: Village Visit
* September 4, 2023, to October 6, 2023: Lab work

### October

* October 1st week: Lab work continued, & Wiki page- refinement was carried out
* October 3, 2023, to October 5, 2023: CAT 2 Examinations
* October 6, 2023: Visited TICEL Biopark
* October 7, 2023: Submitted our Attribution form
* October 10, 2023: Submitted our Judging form and final review of our Wiki page
* October 11, 2023: We completed our Wiki page and it was ready to be submitted
* October 12, 2023: Started working on our project presentation video

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

### **ENGINEERING**

## Laccase

* **Design:** This part consists of a pH-inducible promoter, secretory signal peptide, laccase
  enzyme-coding sequence, a Tandem GS linker, and a reporter gene. The pH-inducible promoter, Asr (which
  stands for acid shock RNA), gets activated in the pH range of 3.5-5.5. With the help of the secretory signal
  peptide, the laccase enzyme is secreted extracellularly. In order to confirm the secretion of the laccase
  enzyme at the laboratory level, we added the reporter gene (CFP) in fusion with the laccase gene using a 13
  Tandem GS linker.
* **Build:** The gene circuit design has been constructed using SnapGene.

  ![](https://static.igem.wiki/teams/4864/wiki/lab/snapgene-1.png)
* **Test:** ABTS assay is a common method to characterize the activity of the enzyme laccase. In this
  assay, ABTS, which is a colorless substrate, is oxidized by laccase, leading to the formation of a
  yellow-green product that can be measured at 420 nm. The absorbance value of the product correlates to the
  activity of laccase. Laccase was added to ABTS, and at regular time intervals, the absorbance was measured,
  and thereby the activity of Laccase was characterized

  ![](https://static.igem.wiki/teams/4864/wiki/lab/laccase-activity.jpg)
  ![](https://static.igem.wiki/teams/4864/wiki/lab/abts.jpg)
* **Learn:** We initially faced issues with the design of the gene circuit, like the distance between RBS
  and the gene of interest, so that gene expression occurs properly. We had discussions with our alumni,
  experts, and figured out the issue, and modified the circuit.

## Cry2Aa

* **Design:** This part consists of a pH-inducible promoter, a Cry2Aa-coding sequence, a Tandem GS
  linker, and a reporter gene. The pH-inducible promoter, Alx (which stands for Alkaline Xylanase), gets
  activated in the pH range above 7. In order to confirm the secretion of the Cry2Aa protein at the laboratory
  level, we added the reporter gene (YFP) in fusion with the laccase gene using a 13 Tandem GS linker.
* **Build:** The gene circuit design has been constructed using SnapGene

  ![](https://static.igem.wiki/teams/4864/wiki/lab/snapgene-2.png)
* **Test:** In this experiment, the plasmid pUC19 carrying the gene of interest- CRY2Aa, was chemically
  synthesized and introduced into competent E. coli bacteria through transformation. Once the transformation
  procedure was completed, the E.coli was cultured, and the transformed colonies were distinguished from the
  non-transformants by inoculating them on an agar plate infused with ampicillin. After completion of
  screening, pUC19 was isolated from E. coli, and the extracted plasmid carrying Cry2AA was then run and
  visualized on the 0.8% agarose gel with a marker. This confirms the presence of the plasmid in E. coli and
  proves its integrity.

  ![](https://static.igem.wiki/teams/4864/wiki/lab/cry.jpg)
* **Learn:**We faced a few issues in the transformation experiment; we had to change our protocol
  according to the experiment and got the results.

## pAsr

* **Design:** This part consists of a pH-inducible promoter and a reporter gene. The pH-inducible promoter,
  Asr (which stands for Acid Shock RNA), gets activated in the pH range below 7. In order to check the
  promoter working, this construct has been designed.
* **Build:** The gene circuit design has been constructed using SnapGene.
* **Test:**
  + Asr promoter (Pasr) is reported to be induced under acidic conditions.
  + It can be used as a reporter when the medium turns acidic. We thus measure the fluorescence intensity
    over a short period of time.
  + We first incubated the bacteria to the log phase (about 2 hours) in Luria-Bertani (LB) medium.
  + We then centrifuged the broth and resuspended the pellet using M9 medium with different pH values (pH
    4, 4.25, 4.5, 4.75, 5, 5.5, 6, and 7; the pH value was adjusted with 1M HCl).
  + We then incubated it in the 96 well plates and measured its fluorescence intensity (absorbance: 485
    nm, excitation: 535 nm) every 3 minutes for 30 minutes.
  + The difference in fluorescence intensity can be observed within 30 minutes.

  ![pasr](https://static.igem.wiki/teams/4864/wiki/lab/pasr.png)

## pAlx

* **Design:** This part consists of a pH-inducible promoter and a reporter gene. The pH-inducible promoter,
  Alx (which stands for Alkaline Xylanase), gets activated in the pH range above 7. In order to check the
  promoter working, this construct has been designed.
* **Build:** The gene circuit design has been constructed using SnapGene.
* **Test:** With this construct, we showed that the alx promoter and riboswitch are able to regulate
  mNeonGreen expression, when pH is shifted to pH 8 and above.
  To do so, we let bacteria grow and then inoculated fresh media with adjusted pH (7, 8, and 8.5) to an OD600 of 0.2.
  After 20 and 40 min, 1 ml of each culture was taken and diluted to the lowest OD600 of the three samples, and
  then fluorescence and OD600 were measured with the plate reader (n = 3).

  ![palx](https://static.igem.wiki/teams/4864/wiki/lab/palx.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## EXPERIMENTS

## Agarose Gel Electrophoresis

#### Materials required

* Electrophoresis apparatus
* Micropipettes
* Eppendorfs

#### Reagents required

* Stock Buffer
* TBE/TAE/TE- 10X / 50X
* Agarose- 0.8% (W/V)
* Ethidium bromide- 10mg/ml

* Tracking Dye
* Glycerol- 30%
* Bromophenol blue- 0.025%

#### Preparation of agarose gel (0.8% concentration for 50ml)

For a gel platform of 10x7cm of thickness 0.6cm (~ 50 ml)

1. Add 0.4g of agarose to a 50ml of 1X TBE (Working Buffer)
2. Heat until the agarose dissolves.
3. Add 5μl of ethidium bromide from a stock when the gel is about 56˚C (final concentration of 0.5μg/ml)

The volume of the well= well width x comb thickness x gel thickness gap

#### Procedure

1. Wash the gel platform thoroughly and wipe with tissue paper.
2. Seal the open sides with adhesive tape.
3. Place the comb on the gel tray in the appropriate place so that a gap of 0.5mm is left between the
   bottom of the comb's teeth and the floor of the gel platform.
4. Pour the warm agarose solution into the gel tray and allow it to solidify.
5. After the gel is completely solidified (30-45 min at room temperature), carefully remove the comb and
   untape, and mount the gel into the electrophoresis tank.
6. Add just enough electrophoresis buffers to cover the gel to a depth of about 1mm.
7. Connect the cords between the electrophoretic tank and the power pack.
8. Mix the 5 μl of the sample with 5 μl of loading dye and load into the slots of the submerged gel using
   a
   disposable micropipette.
9. Fix the initial voltage in the power pack at 50V for the first half an hour and subsequently increase
   the voltage to 150V after 30 minutes.
10. Monitor the movement of DNA with loading dye and switch off the power pack before the dye with DNA
    reaches the lowest point of the gel.
11. Monitor the resolved DNA under a UV Transilluminator at 260nm and infer the result.

## Restriction Digestion

#### Materials Needed

* λ DNA or plasmid DNA
* Restriction enzymes (EcoRI, BamHI)
* 10X restriction enzyme buffer
* Agarose
* Gel loading buffer
* Staining dye
* 50X TAE buffer

#### Protocol for single digestion

1. Take a sterile 0.5ml microfuge tube

2. Add the following reagents in the order listed to a microfuge tube for a 20µl reaction

#### Single Digestion

|  |  |
| --- | --- |
| Sterile DD Water | 15µl |
| Restriction Buffer | 2µl |
| DNA | 2µl |
| Enzyme | 1µl |
| Total | 20µl |

#### Double Digestion

|  |  |
| --- | --- |
| Sterile DD Water | 13µl |
| Restriction Buffer | 2µl |
| DNA | 3µl |
| Enzyme 1 | 1µl |
| Enzyme 2 | 1µl |
| Total | 20µl |

3. Mix it by pipetting and incubate the reaction at the appropriate temperature (37°C).

4. Run the restricted sample on a 1% agarose gel and observe the bands under the transilluminator.

## Ligation

|  |  |
| --- | --- |
| Vector | 3µl |
| Purified PCR fragment | 4µl |
| 5X Ligation Buffer | 6µl |
| T4 DNA Ligase | 1µl |
| NFW (Nuclease Free Water) | Upto 29µl |

Incubate @ 22°C for 1 hour. For maximum yield of useful recombinants, the reaction time can be extended to
overnight @ 4°C.

## PCR

#### PCR Reaction Mixture

|  |  |
| --- | --- |
| Master Mix | 12.5 |
| Forward Primer | 1.0 |
| Reverse Primer | 1.0 |
| Template | 2.0 |
| Nuclease Free Water (NFW) | 8.5 |
| Total | 25.0 |

#### Cyclic Conditions

The reaction mixture was denatured at 94°C for 5 min. Then PCR was run for 30 cycles at 94°C for 2 min, and
annealing and extension temperature of 55°C for 1 min. The sample was then heated at 72°C for 1 min.
Extension
and final extension reaction are at 72°C for 7 mins. Finally, the product will be checked with 1% Agarose
Gel
electrophoresis with 1Kb DNA ladder. After electrophoresis, it is viewed under UV light.

## Competent Cell Preparation

#### Materials Required

* Detergent-free, sterile glassware, and plasticware
* Table-top OD600nm spectrophotometer
* SOB
* CCMB Buffer

#### Procedure

1. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. Use
   the
   "cell culture" function on the Nanodrop to determine OD value. OD value = 600nm Abs reading x 10.
2. Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat-bottom centrifuge bottles as
   needed.
3. Transfer the culture to the flat-bottom centrifuge tubes. Weigh and balance the tubes using a scale.
4. Centrifuge at 3000g at 4°C for 10 minutes in a flat-bottom centrifuge bottle.
5. Decant supernatant into the waste receptacle and bleach before pouring down the drain.
6. Gently resuspend in 80 ml of ice-cold CCMB80 buffer.
7. Incubate on ice for 20 minutes.
8. Centrifuge again at 3000g at 4°C. Decant supernatant into the waste receptacle, and bleach before
   pouring
   down the drain.
9. Resuspend the cell pellet in 10 ml of ice-cold CCMB80 buffer.
10. Use Nanodrop to measure the OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
11. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
12. Incubate on ice for 20 minutes. Prepare for aliquoting.
13. Aliquot into chilled 2ml microcentrifuge tubes or 50 μl into chilled microtiter plates.
14. Store at -80°C indefinitely.
15. Test competence by measuring O.D.
16. Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by
    about
    3x the first time, and about 6x total after several freeze/thaw cycles.

## Transformation

#### Procedure

1. Prepare 50 μl of competent cells in Eppendorf tubes.
2. Add 1 μl of plasmids to each Eppendorf tube containing competent cells.
3. Mix the contents well and incubate on ice for 30 minutes.
4. Provide heat shock by placing the tubes at 42ºC for 2 minutes. Immediately, snap-chill the tubes in ice
   and
   incubate for an additional 10 minutes.
5. Add 200 μl of LB medium to each tube and incubate at 37ºC for 1-2 hours.
6. After incubation, spread-plate the culture onto the respective antibiotic-containing plates.
7. Incubate the plates for 48 hours at 37ºC and check for transformation efficiency.

## Elution

#### Materials required

* Elution buffer (0.5 M sodium acetate (pH 7) and 1 mM EDTA (pH 8))
* 95% ethanol
* 80% ethanol
* TE buffer (10x) (pH 8.0)
* Gel fragment containing the DNA

#### Procedure

1. Excise the region of the gel containing the required DNA band using a scalpel.
2. Add elution buffer to the gel slice until the level of the buffer is a few mm above the level of the
   excised
   gel band.
3. Heat the solution in a water bath at 65°C until the agarose completely melts.
4. Fast-freeze by placing in a -80°C freezer for 10-15 minutes.
5. Immediately thaw the solution by centrifuging for 10 minutes.
6. The supernatant is transferred to a new tube.
7. Add elution buffer again to the pellet and repeat steps 3-6.
8. Accumulate the supernatants and add an equal volume of 1-butanol.
9. Rock the mixture for 15 minutes to remove the EtBr completely from the gel.
10. Discard the supernatant and repeat steps 8 and 9 2-3 times.

## ABTS Assay

#### Procedure

1. The assay mixture contained 450 μL of 1 mM ABTS, 500 μL of 0.1 M acetate buffer (pH 4.0) along with 50
   μL
   of enzyme at a temperature of 20 °C.
2. This mixture was tilted in the cuvette before taking the absorbance reading.
3. The complete reaction took place in the cuvette and nowhere else.
4. The duration of the reaction was not more than 60 s.

## Promoter Characterization

## pAlx - 96 Well Plate Reader Experiment

* With this construct, we showed that the alx promoter and riboswitch are able to regulate mNeonGreen expression, when pH is shifted to pH 8 and above.
* To do so, we let bacteria grow and then inoculated fresh media with adjusted pH (7, 8, and 8.5) to an OD600 of 0.2.
* After 20 and 40 min, 1 ml of each culture was taken and diluted to the lowest OD600 of the three samples, and then fluorescence and OD600 were measured with the plate reader (n = 3).

## pAsr - 96 Well Plate Reader Experiment

* Asr promoter (Pasr) is reported to be induced under acidic conditions.
* It can be used as a reporter when the medium turns acidic. We thus measure the fluorescence intensity over a short period of time.
* We first incubated the bacteria to the log phase (about 2 hours) in Luria-Bertani (LB) medium.
* We then centrifuged the broth and resuspended the pellet using M9 medium with different pH values (pH 4, 4.25, 4.5, 4.75, 5, 5.5, 6, and 7; the pH value was adjusted with 1M HCl).
* We then incubated it in the 96 well plates and measured its fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) every 3 minutes for 30 minutes.
* The difference in fluorescence intensity can be observed within 30 minutes.

{% endblock %}

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

### RESULTS

## Transformation of PUC19 in E. coli

In this experiment, the plasmid pUC19 carrying the gene of interest- CRY2Aa, was chemically synthesized and introduced into competent E. coli bacteria through transformation. Once the transformation procedure was completed, the E.coli was cultured and the transformed colonies were distinguished from the non-transformants by inoculating them on an agar plate infused with ampicillin.

![bw-screen](https://static.igem.wiki/teams/4864/wiki/lab/bw-screen.jpg)

Figure 01:Transformed white colonies on ampicillin medium

## Isolation of PUC19 containing Cry2Aa from transformed E. coli and visualization on gel electrophoresis

After completion of screening, pUC19 was isolated from E. coli, and the extracted plasmid carrying Cry2Aa was then run and visualized on the 0.8% agarose gel with a marker. This confirms the presence of the plasmid in E. coli and proves its integrity.

![puc](https://static.igem.wiki/teams/4864/wiki/lab/whatsapp-image-2023-10-11-at-07-07-42-aef29fcc.jpg)

Figure 02: Isolated PUC19 with Cry2Aa

## Elution of PUC19 from the gel and restriction digestion to restrict the gene of interest

Once gel electrophoresis was performed, the isolated plasmid pUC19 was eluted from the agarose gel. Subsequent digestion by restriction enzymes BamHI and NcoI resulted in the cleavage of the Cry2Aa gene. The separated gene was then subjected to Agarose gel electrophoresis, alongside a 1000-kb DNA ladder, and visualized through a UV-gel Transilluminator.

![cry](https://static.igem.wiki/teams/4864/wiki/lab/cry.jpg)

Figure 03: Digested Cry2Aa with NcoI and BamHI

## ABTS assay

ABTS assay is a common method to characterize the activity of the enzyme laccase. In this assay, ABTS which is a colorless substrate, is oxidized by laccase, leading to the formation of a yellow-green product which can be measured at 420 nm. The absorbance value of the product correlates to the activity of laccase. Laccase was added to ABTS, and at regular time intervals, the absorbance was measured, and thereby the activity of Laccase was characterized.

![abts](https://static.igem.wiki/teams/4864/wiki/lab/abts.jpg)

Figure 04: ABTS Assay

![laccase activity](https://static.igem.wiki/teams/4864/wiki/lab/laccase-activity.jpg)

Figure 05: Enzymatic activity of laccase under various substrate concentrations.
The laccase enzyme was assayed in the presence of substrate ABTS, and enzymatic oxidation of ABTS by laccase was observed at various time intervals continuously under standard assay conditions.

{% endblock %}