iGEM 2023 — Team ict-mumbai

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{% block title %}Notebook{% endblock %}
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## List of meetings:

| DATE | Agenda |
| --- | --- |
| 9/4/2023 | Fundraising |
| 19/4/2023 | Fundraising |
| 20/4/2023 | iGEM Team recruitment orientation |
| 21/4/2023 | Script for orientation |
| 25/4/2023 | Discussion on when to begin lab activities and IHP |
| 2/5/2023 | Lab equipment |
| 4/5/2023 | Introductory meeting, assigning task to every individual, set deadlines and brainstorming ideas. |
| 6/5/2023 | Brainstorming Ideas for Educational Activities |
| 8/5/2023 | Setting a Timeline for Finalised Educational Activities and IHP |
| 15/5/2023 | Preparations begin for conducting Educational Activities including contacting potential institutions, schools and people |
| 22/5/2023 | Discussing about Responses from the contacted institutions and people |
| 26/5/2023 | Designing Primers; Brainstorming educational activities and discussing plan of action for the confirmed ones |
| 29/5/2023 | Designing Primers |
| 30/5/2023 | Approaching Dr Anuradha Borkar and Dr Rosemarie de Souza |
| 31/5/2023 | Lab work briefing and start |
| 5/6/2023 | Discussion about AIIM |
| 6/6/2023 | Meeting with SMSR for volunteer work of Synbio workshop |
| 7/6/2023 | Discussion with Educational Activities team about Synbio workshop |
| 8/6/2023 | Meeting with Dietician Charolette Jhakaria, wet lab team PCR troubleshooting meeting |
| 9/6/2023 | Discussing plan of action for the SynBio Workshop |
| 10/6/2023 | Meeting with other iGEM teams regarding finalization of Date and Venue of AIIM 2023 |
| 11/6/2023 | Preparation for Synbio workshop; Ketto link Login |
| 12/6/2023 | Preparation For Synbio Workshop |
| 13/6/2023 | Synbio Workshop at KJ Somaiya |
| 14/6/2023 | Synbio Workshop at KJ Somaiya |
| 15/6/2023 | Synbio Workshop at KJ Somaiya |
| 16/6/2023 | Wet Lab troubleshooting |
| 17/6/2023 | Preparing project description; Discussion with IISER-Pune about conduction Yeast Biosafety Workshop |
| 19/6/2023 | Project description; Educational Activities Team Discussion about speaker for Biosafety Workshop |
| 20/6/2023 | Wet lab troubleshooting |
| 21/6/2023 | Project description |
| 23/6/2023 | Approaching nutritionist Ishwa Desai, wet lab team meeting for troubleshooting |
| 26/4/2023 | Setting timelines about educational activities and brainstorming for the following ones |
| 27/6/2023 | Contacting potential people for collaborating in conduction of educational activites |
| 28/6/2023 | Contacting potential people for collaborating in conduction of educational activites |
| 29/6/2023 | Discussing about received responses and planning for activities |
| 30/6/2023 | Wet lab team – troubleshooting; meeting to discuss approaching industries |
| 2/7/2023 | Meeting to finalize the course for the next activity and form a tentative schedule for further activities |
| 6/7/2023 | Project description, write-ups for PR team, summarizing past year iGEM projects related to our topic |
| 7/7/2023 | Wet lab team meeting with SMSR |
| 9/7/2023 | Wiki page designing for the project and project description |
| 10/7/2023 | Discussing results of SOE PCR |
| 11/7/2023 | Discussing about education activities |
| 12/7/2023 | Discussing about educational activities |
| 15/7/2023 | Meeting with Mr. Iliyaz from Bristol bakery |
| 24/7/2023 | AIIM |
| 31/7/2023 | AIIM |
| 1/8/2023 | Finalising details with school about conduction of DNA Cloning Seminar |
| 3/8/2023 | Discussing about educational activities |
| 4/8/2023 | Wet lab team meeting with SMSR |
| 6/8/2023 | Meeting with Dr Rekha Singhal; more policy makers to approach |
| 7/8/2023 | Preparation for Basic of DNA Cloning Seminar |
| 9/8/2023 | IHP – meeting with a policymaker; Basics of DNA Cloning Seminar at Don Bosco High School |
| 11/8/2023 | Requirement of funds |
| 14/8/2023 | Wiki team meeting |
| 15/8/2023 | Conducted Biosafety Webinar Part II with speaker Dr Vinod Jyothikumar |
| 17/8/2023 | Wet Lab team meeting with SMSR |
| 18/8/2023 | Ketto link publicity |
| 29/8/2023 | Funds expenditure estimates |
| 31/8/2023 | Wet lab team meeting with SMSR |
| 2/9/2023 | Wet lab team meeting with SMSR |
| 6/9/2023 | Meeting with Educational Activities team to plan the activities that’ll follow |
| 7/9/2023 | Form release for participation in Bread Making Activity |
| 9/9/2023 | Selection of Participants for Bread Making Activity and informing them to arrange the needful |
| 11/9/2023 | Wiki team meeting |
| 19/9/2023 | Preparation for Local Bread Making Activity |
| 20/9/2023 | Wet lab team meeting with SMSR; Educational activities team meeting |
| 21/9/2023 | Conducting Local Bread Making Educational Activity |
| 22/9/2023 | Contacting concerned people and their responses about educational activity |
| 27/9/2023 | Wet lab team meeting with SMSR |
| 30/9/2023 | Wet lab team meeting with SMSR; Preparation of educational activity |
| 1/10/2023 | Conduction of educational activity with Underprivileged Kids |
| 3/10/2023 | Wet lab team meeting with SMSR |
| 5/ 10/2023 | Wet lab team meeting with SMSR |
| 7/10/2023 | Wet lab team meeting with SMSR |
| 9/10/2023 | Wet lab team meeting with SMSR |

## Lab Notebook:

Our experiments in the lab started the first week of June and have lasted till October. This notebook contains the account of all our highs and lows, the frustrations and the wins for the other iGEM teams and judges to go through.
Due to an incredibly late start, we have fallen short of achieving everything we envisioned but nonetheless we are proud of what we have managed to do. We hope the reader finds the lab notebook insightful.

[Click here to know more](https://onedrive.live.com/view.aspx?resid=9500C49FD7AFBB73%213493&authkey=!ADSOoM7nuX-aba0)
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## Engineering

Our aim was to use the BioBrick format for the plasmids pRS426 and YIplac204 as well as the inserts, so as to produce phytase enzyme in the host organism, which we chose to be Saccharomyces cerevisiae.

![Animation]({{url_for('static', filename='picture.jpeg')}})

Amplification of the genes
Amplification of the plasmid
Restriction-digestion (RD)
Adding into yeast cells
Phytate content estimation

## Amplification of the genes

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:** Amplifying the genes using a high-fidelity polymerase in a normal PCR reaction so that we could further perform restriction-digestion and then ligate them to our plasmid (pRS426GPD)

**Build:** Set up the PCR reaction

**Test:** Gel electrophoresis showed bands of much smaller length than that of the gene of interest.

**Learn:** PCR reaction was not successful.

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 2

**Design:** Since the high-fidelity enzyme (phusion) did not work, we decided to use Q5 and a combination of Taq and Pfu polymerase in varying proportions for the amplification.

**Build:** Set up the PCR reaction

**Test:** Gel electrophoresis did not show bands indicating desired length.

**Learn:** None of the afore mentioned polymerases worked. PCR was unsuccessful.

###### After multiple tries at trouble-shooting for 3 weeks, we arrived at the conclusion that the polymerases we were using were degrading our primers very quickly because they had longer “tails”

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:** We then decided on using a long-range polymerase for amplification of the genes.

**Build:** Set up the PCR

**Test:** Gel electrophoresis of the purified PCR product using a long-range polymerase showed bands of desirable lengths.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . **Learn:** **A long-range polymerase showed positive results** for our PCR

![]({{url_for('static', filename='demo.jpeg')}})

## Amplification of the plasmid fragments and joining the fragments using SOE PCR

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:** Since we had to design primers such that we could avoid the PstI site in our plasmid, we decided to amplify the individual fragments and then use splice over extension (SOE) PCR to join the fragments together. We will also be requiring an equimolar mixture of the fragments for performing SOE PCR.

**Build:** Set up the PCR reaction for amplification; quantify the fragment mixtures; set up SOE PCR.

**Test:** Bands at desirable lengths obtained for the amplification; No bands visible after the setting up of SOE PCR.

**Learn:** SOE PCR was not successful and needed to be troubleshooted.

###### After multiple changes in our protocol, our SOE PCR was still unsuccessful! To show proof of concept, we decided to use different restriction sites for the amplification of the genes to introduce into the plasmid.

## Restriction-digestion (RD) of the inserts and the plasmid

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:**Producing sticky ends using REases to facilitate ligation of the fragments.

**Build:** Set up RD followed by gel purification

**Test:** Gel electrophoresis showed bands at desirable length

**Learn:** RD was successful

###### Ligation was set up for these fragments and E. coli transformation was carried out:

![](https://drive.google.com/uc?export=view&id=1TvCQJ9KKUKsfLJjPTUNfmN8eNtzMhTY3)
![](https://drive.google.com/uc?export=view&id=1M92e2iugOGbqVJXgEzyvLql1AtUSJgMd)

Image Credits:Team ICT-Mumbai 2022

## Adding the recombinant plasmid into yeast cells

![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:**Isolate the recombinant plasmid from the transformed E. coli and insert into Saccharomyces cerevisiae

**Build:** Yeast transformation protocol was carried out and the culture was plated on medium – agar + YNB + complete synthetic medium w/o URA + glucose

**Test:** Colonies were observed on the second day after plating. Observing under the microscope confirmed it was yeast.

**Learn:** Yeast transformation was successful

![](https://drive.google.com/uc?export=view&id=11D5WkRecBn_lpeTKKAJbsznaGWoBjUj9)

## Phytate content estimation

ś![](https://static.igem.wiki/teams/4692/wiki/eng1.png)

#### 1

**Design:**We decided to estimate the decrease in phytate content in a given sample by measuring one of the products formed after the breakdown of phytic acid upon the addition of our enzyme after intervals of half hour and 1h. We decided to use the Fiske-Subarrow method for the estimation of free inorganic phosphate in the reaction mixture after those stipulated time intervals.

**Build:** Carried out the protocol for the Fiske-Subarrow method, a colorimetric method for the estimation of inorganic phosphate.

**Test:** While preparation of the samples for the estimation, we observed our blank was also showing colour upon the addition of the reagent.

. . . . . . . . . . . . . . . . . . . . .**Learn:** **Phytic acid also gives positive results for the Fiske-Subbarrow method.** An alternate method for the estimation of phytate or modifications to the existing method required.

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#### The production of phytase from phytate through yeast cell modification has the potential to positively impact both bakeries in India and the health of the general public:

![Image]({{url_for('static', filename='picture.jpeg')}})

## 1. End-product:

Our intended end-product can be a wet yeast for direct use in smaller bakeries or dried yeast to be added for use for large scale bakeries; it can also be added into premixes as suggested by dietician Charolette Jhakaria, for traditional dishes like dosa or idli.

## 2. Public Health:

Nutrient Availability: Phytase can increase the bioavailability of essential minerals like phosphorus, iron, and zinc in baked goods, potentially addressing micronutrient deficiencies.

Healthier Diet: The enriched bread may contribute to a more nutritious diet, potentially benefiting the general public's health in the long run.

Overall, the yeast cell modification for phytase production has the potential to enhance bakery products and improve the nutritional content of daily diets, potentially benefiting both businesses and public health in India.

Enhancing the nutritional quality of livestock feed is a promising initiative with the potential to benefit consumers worldwide. By fortifying animal feed, we can ensure that the livestock raised for human consumption receive essential nutrients, ultimately improving the nutritional value of the meat and dairy products derived from them.

To adhere to regulatory guidelines, such as those outlined by the OECD, it is imperative to conduct rigorous assessments. This includes evaluating genotoxicity and health hazards associated with feed preparation. Additionally, a critical aspect is investigating the digestibility of supplemented feed. If incomplete digestion occurs, it is essential to demonstrate that any resulting metabolites are non-toxic and non-allergenic.

The outcome of these assessments will provide crucial insights into the key nutrients and potential toxicants present after supplementation, ensuring that the fortified livestock feed remains safe, nutritious, and beneficial for both animals and consumers alike.

#### Rules and regulations:

In India, the FSSAI is the food regulations authority which states the following prerequisites for the prior approval for manufacture, storage, distribution, sale and import etc.— (1) No person shall manufacture, pack, store, sell, market or otherwise distribute or import any food or food ingredient produced from GMOs, except with the prior approval of the Food Authority. (2) The provisions of these regulations are in addition to, and not in derogation of, any other rules or regulations made under the Act.

Procedure for grant of prior approval— (1) The food business operator (manufacturer/importer) of a GM-food shall submit an application as per the format prescribed by the Food Authority along with necessary documents and fee to the Food Authority. (2) If the application is for a GMO referred to as in clause (a) of sub-regulation (2) of regulation 1, clearance from Genetic Engineering Appraisal Committee (GEAC), the national competent authority for the environmental risk assessment shall be submitted to the Food Authority.

##### Our product will have to first receive clearance from GEAC.

If our intended final product receives a ‘Unique Identifier Nine-digit Code’ of the Organisation for Economic Cooperation and Development (OECD) that is approved for food use by the FSSAI, it will require no further approvals.

Our conversation with a policymaker, Dr Kapila Mehta

![Kapila Mehta](https://drive.google.com/uc?export=view&id=1UJTW7iuuDb4UzzTGqMLuRqN87YMbYTTb)

## 3. Implementation in bakery industry:

To overcome challenges related to the effects of fermentation on phytase and achieve desired outcomes, industries may need to implement various process optimizations. These optimizations can vary depending on the specific goals and challenges faced by each industry.

Here are some potential process optimizations:

Fermentation Conditions: Adjust fermentation conditions such as temperature, pH, and oxygen levels to create an environment conducive to phytase activity. This may involve optimizing these parameters for the specific microorganisms used.

Fermentation Duration: Optimize the duration of fermentation to balance the desired increase in phytase activity with the risk of enzyme inactivation over time. Monitoring enzyme activity throughout the process is crucial.

Enzyme Production: Develop strategies to enhance the production of phytase enzymes during fermentation. This might involve using inducers that stimulate enzyme production.

Co-Fermentation: Explore co-fermentation with microorganisms that produce organic acids or other compounds that can enhance phytase activity. This can create a more favourable environment for enzyme function.

Monitoring and Control: Implement robust monitoring and control systems to continuously assess phytase activity and adjust fermentation conditions in real-time. This can help maintain optimal enzyme activity throughout the process.

###### Impact on Bakeries in India:

- Improved Dough Quality: Phytase can enhance dough quality, leading to better texture and volume in baked goods like bread.

- Cost Savings: It can reduce the need for costly additives, resulting in more cost-effective bakery operations along with the prospects of employing simultaneous fermentation.

- Enhanced Product Portfolio: Bakeries may diversify their product offerings with improved quality, potentially attracting more customers.

## 4. Future iGEM teams:

Future iGEM teams can play a pivotal role in fortifying essential nutrients in both human and animal food products by building upon our work as a foundational starting point. Our recombinant plasmid can be used to introduce genes for the synthesis of other nutrients like vitamins, to be transformed into microorganisms efficiently. iGEM teams can use this platform to engineer microorganisms for nutrient production and enhance the nutritional value of everyday foods through collaboration and innovative solutions. Our shared goal is to address global health challenges related to nutrition and improve public health by fortifying commonly consumed foods.

![Image](https://drive.google.com/uc?export=view&id=1LFjA_yqj5xaDU_S0Ge1K20aIlMAkTRuF)

## 5. Safety:

Our project involves genetically modifying the yeast Saccharomyces cerevisiae to express the enzyme phytase. S. ce revisiae is classified as GRAS (generally regarded as safe), and the enzyme that is being overexpressed is not toxic.

We are using an auxotrophic marker (URA3) for transforming S. cerevisiae with an episomal plasmid containing the phytase gene. For genome integration too, an auxotrophic marker (TRP1) will be used. No antibiotic markers will be used.

However, as the plasmids we are using are E. coli-S. cerevisiae shuttle vectors, the bla gene (ampicillin antibiotic marker) is present in the vector. Integration of a linear DNA fragment containing just the expression cassette and an auxotrophic marker in the yeast genome is an approach which we plan to take once we have established proof of concept.

As the constructed yeast strain will be used for leavening breads, which are baked before consumption, we do not expect release of the modified organism in the environment.

References:
[1] 50374/2022/REGULATION-FSSAI
[2] OECD’s Report of the task force for the safety of novel foods and feeds
[3] C. Frontela, G. Ros, C. Martínez, Phytic acid content and “in vitro” iron, calcium and zinc bioavailability in bakery products: The effect of processing, Journal of Cereal Science, Volume 54, Issue 1, 2011, Pages 173-179, ISSN 0733-5210, https://doi.org/10.1016/j.jcs.2011.02.015.
[4] Nuobariene, L., Arneborg, N., & Hansen, Å. S. (2014). Phytase active yeasts isolated from bakery sourdoughs. In 9th Baltic Conference on Food Science and Technology "Food for Consumer Well-Being": FOODBALT 2014 - conference proceedings (pp. 223-227). Latvia University of Agriculture. Baltic Conference on Food Science and Technology

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Our proof of concept experiment consisted of experimentally determining extent of phytate breakdown in dough prepared using a millets, which have a high phytate content.
We used the method described by Heinonen and Lahti (Anal Biochem 1981 May 15;113(2):313-7. doi: 10.1016/0003-2697(81)90082-8) to determine phytate breakdown. This method is used to estimate free inorganic phosphate, which is released when phytate is broken down. Unlike the well-known Fiske-Subbarow method of inorganic phosphate determination, phytate does not cause interference in the method of Heinonen and Lahti.
We constructed a standard curve using KH2PO4 to establish the relationship between Pi and absorbance at 390 nm.

![](https://drive.google.com/uc?export=view&id=12yHYF68mzpXszDJekRe-1OJgTnCV7UJQ)

Using the equation determined from the above experiment, we determined the free Pi content of sorghum (locally known as jowar, a staple diet in rural areas of Maharashtra and Telangana states) and refined wheat flour.

![](https://drive.google.com/uc?export=view&id=12ZwfgOJuV4qo275EQlCFnd7G7C40esIG)

We then proceeded to ferment dough using our constructed yeast strains to determine the extent of Pi liberated.
Dough was prepared using one part each of sorghum and refined wheat flour. The yeast strains were used to ferment the flour for four hours. Samples of the rising dough were taken and concentration of free Pi in the dough was determined.
![](https://drive.google.com/uc?export=view&id=1xsi4LosocgjZXit3APXz2fc_3Q2ENZFl)

As can be seen from the above table, dough fermented by strains expressing Aj and Ec phytases had 10 and 23 percent more Pi as compared to dough fermented by the control strain (P=0.005). This demonstrates that phytase breakdown was significantly higher when phytase expressing strains are used to ferment the dough.

## We baked the dough to make bread, and voila!

![](https://drive.google.com/uc?export=view&id=1EVZ830vyOxaAgBl9IA3c4_isHB6vYl7y)
![](https://drive.google.com/uc?export=view&id=1xXbo3aAESwp17sGbeTRIRKUGWHZfQPx4)
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