iGEM 2019 — Team Bioriidl_Somaiya

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# Team:Bioriidl Somaiya/Notebook

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[HOME](https://2019.igem.org/Team%3ABioriidl_Somaiya)

TEAM

[Team Members](https://2019.igem.org/Team%3ABioriidl_Somaiya/Team)
[Collaborations](https://2019.igem.org/Team%3ABioriidl_Somaiya/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ABioriidl_Somaiya/Description)
[Design](https://2019.igem.org/Team%3ABioriidl_Somaiya/Design)
[Experiments](https://2019.igem.org/Team%3ABioriidl_Somaiya/Experiments)
[Notebook](https://2019.igem.org/Team%3ABioriidl_Somaiya/Notebook)
[Contribution](https://2019.igem.org/Team%3ABioriidl_Somaiya/Contribution)
[Results](https://2019.igem.org/Team%3ABioriidl_Somaiya/Results)
[Demonstrate](https://2019.igem.org/Team%3ABioriidl_Somaiya/Demonstrate)
[Improve](https://2019.igem.org/Team%3ABioriidl_Somaiya/Improve)
[Attributions](https://2019.igem.org/Team%3ABioriidl_Somaiya/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ABioriidl_Somaiya/Parts)
[Basic Parts](https://2019.igem.org/Team%3ABioriidl_Somaiya/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ABioriidl_Somaiya/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ABioriidl_Somaiya/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ABioriidl_Somaiya/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ABioriidl_Somaiya/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ABioriidl_Somaiya/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ABioriidl_Somaiya/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ABioriidl_Somaiya/Hardware)
[Measurement](https://2019.igem.org/Team%3ABioriidl_Somaiya/Measurement)
[Model](https://2019.igem.org/Team%3ABioriidl_Somaiya/Model)
[Plant](https://2019.igem.org/Team%3ABioriidl_Somaiya/Plant)
[Software](https://2019.igem.org/Team%3ABioriidl_Somaiya/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Bioriidl_Somaiya)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:Bioriidl Somaiya/Experiments

Experiments

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* Team
  + Team Members
  + Collaboration
* Project
  + Description
  + Design
  + Experiments
  + Notebook
  + Contributions
  + Results
  + Demonstration
  + Improvements
* Parts
  + Parts Overview
  + Basic Parts
  + Composite Parts
  + Parts Collection
* Safety
* Human Practices
  + Human Practices
  + Education and Engagement
* Awards
  + Entrepreneurship
  + Hardware
  + Software
* Judging Form

# Experiments

# Experiment No. 1

## Testing the efficiency of nichrome wire

* Aim: To check whether the current passed through
  nichrome wire increase the temperature.
* Result: The average time required for a 10 °C rise
  was determined to be 4.2 seconds. Thus, 0.56sec for a 1 °C rise in
  temperature.

Learn More

# Experiment No. 2

## Using nichrome wire

* Aim: To determine the time required for the water to
  raise the temperature by 1 °C using nichrome loop.
* Result: Thus, it takes 1mins 12sec to raise the
  temperature from 25 °C to 100 °C, so 0.96sec is taken to rise 1 °C
  in temperature for water.

Learn More

# Experiment No. 3

## Sterilize NA media by direct heating using nichrome wire

* Aim: To determine the time required for the media to
  raise the temperature by 1 °C.
* Result: Thus it takes 45 seconds to raise the
  temperature of the medium from 25 °C to 100 °C.

Learn More

# Experiment No. 4

## Sterilize media by indirect heating using oil along with nichrome wire (wrapped around the flask containing oil)

* Aim: To determine the time required to raise the
  temperature of the media from 25 °C to 100°C using a nichrome wire
  tied around a flask containing oil which has the medium immersed
  into it.
* Result: This method is not as efficient.

Learn More

# Experiment No. 5

## Sterilize media by indirect heating using oil with nichrome wire wrapped around the tube and dipped in oil.

* Aim: To determine the time required to raise the
  temperature of water from 25 °C to 100 °C using nichrome wire
  directly wrapped around tube and dipped in flask containing oil
  for heat preservation.
* Result: This method was ruled out.

Learn More

# Experiment No. 6

## Sterilize media by indirect sterilization using powder ink

* Aim: To determine the time required to raise the
  temperature of water from 25 °C to 100 °C by using ink powder
  particles (magnetic) by holding over induction coils.
* Result: Method ruled out.

Learn More

# Experiment No. 7

## Viscosity checking of medium that can be used

* Aim: To check the time required for water, and
  various media to flow through Ostwald's viscometer.
* Result: Liquid media passed easily through the tiny
  capillary whereas heated agar was very difficult to pass. Hence
  tubing dimensions could be decided for further manufacture.

Learn More

# Experiment No. 8

## Developing UV Chamber

* Aim: Circuit assembly inside an acrylic chamber with
  holding space for petri dishes in the centre. UV- C tubes
  installed inside the chamber on top and bottom to evenly irradiate
  the plates.

Learn More

# Experiment No. 9

## Sterilize using UV-C germicidal tube

* Aim: To determine effectivity of the manufactured UVC
  chamber by exposing plates with plain unsterilized medium to UV
  radiation for varied amounts of time and incubation of plates and
  enumeration.
* Result: 8 minutes of exposure to UV was enough for
  killing microbial population in media.

Learn More

# Experiment No. 10

## Media preparation

* Aim: Preparation of 250 ml Sabourauds agar and 24
  petri plates and set it for autoclaving.

Learn More

# Experiment No. 11

## Endophyte experiment

* Aim: Isolation of endophytes from Neem (Azadirachta
  indica) and Tulsi (Ocimum tenuiflorum).
* Result: Presence of Endophytes have been observed and
  they have been screened for further use.

Learn More

# Experiment No. 12

## Subculturing of endophytes to obtain a pure-culture

Learn More

# Experiment No. 13

## Replating of colonies

* Aim: Replating and segregation of colonies.

Learn More

# Experiment No. 14

## Isolation of grown colonies

* Aim: Isolation of various different colonies found on
  sabourauds plates which had plant parts.
* Result: There were pure culture growth of colonies
  with no other colony presence.

Learn More

# Experiment No. 15

## Checking growth

* Observations: Most plates showed pure culture
  characteristics. These colonies were chosen.

Learn More

# Experiment No. 16

## Mass culture

* Aim: Making large amounts of the chosen culture.

Learn More

# Experiment No. 17

## CAD modeling of U.V. chamber using AutoCAD Software

Learn More

# Experiment No. 18

## New UV - C chamber manufacture

Learn More

# Experiment No. 19

## Testing UVC chamber using plates

* Aim: To test microbicidal effect of the new UVC
  assembly by using plates. Preparation of NB and using 9 plates to
  test for U.V. (for 2,4,6,8,10mins closed plate, 5mins closed,
  5mins open).
* Result: 5 minutes were enough to sterilize the media
  under this new chamber.

Learn More

# Experiment No. 20

## Spiral Tube experiments

Learn More

# Experiment No. 21

## Exploration of desired genes

* Aim: To select a protein/compound secreted by
  organism that has killing/ inhibitory activity on contaminating
  organisms.

Learn More

# Experiment No. 22

## Model testing using various media

* Aim: To see the effect of UV on organisms growing on
  different media.
* Result: MacConkey media and LB media showed minimum
  growth of organisms and Mueller Hinton agar showed very scanty
  growth of contaminants.Maximum growth was found in Nutrient agar.

Learn More

# Experiment No. 23

## Prototype building

* Aim: To build a prototype of our experiment using CAD
  model.

Learn More

Bioriidl © Copyright 2019. All rights reserved.

×

## Testing the efficiency of nichrome wire

Date: 6th April 2019

×

## Using nichrome wire

Date: 12th April 2019

Time: 2:00 PM - 6:00 PM

Total number of attendees: 5

Number of male attendees: 2

Number of female attendees: 3

Ashtad, Shreya, Mihir, and Apeksha

Aim - To determine the time required for the water to raise the temperature by 1 °C using nichrome loop.

Protocol -

Step 1: - Firstly a nichrome wire is twisted around a test tube with the help of captain tap (As this tap can sustain high and current).

Step 2: - 5ml of water is taken into the test tube.

Step 3: - Two sprung metal clips were attached on the ends of nichrome wire and DC power supply giving 3.2A and 32 Volt was passed.

Step 4: - Repeat the above steps for 3-4 times for the average result.

Observation - The temperature of water rises from 25 °C to 100 °C in an average time of 2mins 46secs.

The result - Thus, it takes 1mins 12sec to raise the temperature from 25 °C to 100 °C, so 0.96sec is taken to rise 1 °C in temperature for water.

×

## Sterilize NA media by direct heating using nichrome wire

Date: 15th - 17th April 2019

Time: 2:00 PM - 6:00 PM

Total number of attendees: 4

Number of male attendees: 2

Number of female attendees: 2

Ashtad, Shreya, Mihir, and Apeksha

Aim - To determine the time required for the media to raise the temperature by 1 °C.

Protocol -

Step 1: - Firstly a nichrome wire is twisted around a test tube with the help of captain tape (As this tape can sustain high heat and current).

Step 2: - 5ml of this is taken into the test tube.

Step 3: - Two sprung metal clips were attached on the ends of nichrome wire and DC power supply giving 3.2A and 32 Volt was passed.

Step 4: - Using a thermometer for measuring, increase in temperature of the medium was noted and tabulated.

Observation- The temperature of the water in the tube raises to 100 °C in 45 seconds.

Result- Thus it takes 45 seconds to raise the temperature of the medium from 25 °C to 100 °C.

×

## Sterilize media by indirect heating using oil along with nichrome wire (wrapped around the flask containing oil)

Date: 22nd - 23rd April 2019

Time: 2:00 PM - 6:00 PM

Total number of attendees: 4

Number of male attendees: 2

Number of female attendees: 2

Ashtad, Shreya, Mihir, and Apeksha

Aim - To determine the time required to raise the temperature of the media from 25 °C to 100°C using a nichrome wire tied around a flask containing oil which has the medium immersed into it.

Protocol -

Step 1 - First the nichrome wire is wrapped around the flask which is filled with oil.

Step 2 - 5ml water is taken in a tube and immersed in the oil

Step 3 - Two sprung metal clips were attached on the ends of nichrome wire and DC power supply giving 3.2A and 32 Volt was passed.

Step 4 - Using a thermometer for measuring, increase in temperature of the medium was noted and tabulated.

Step 5 - The reading were compared with that of only nichrome wire

Observation- The temperature of the water raised from 25 °C to 100 °C in 2 mins 20 seconds.

Result- This method is not as efficient.

×

## Sterilize media by indirect heating using oil with nichrome wire wrapped around the tube and dipped in oil

Date- 23rd April 2019

Time- 2:00 pm- 6:00 pm

 Ashtad, Shreya, Mihir and Apeksha

 Aim - To determine the time required to raise the temperature of water from 25 °C to 100 °C using nichrome wire directly wrapped around tube and dipped in flask containing oil for heat preservation.

 Protocol -

 Step 1 - The nichrome wire is wrapped around the tube containing 5 ml water and dipped in flask filled with oil.

 Step 2 - Two sprung metal clips were attached on the ends of nichrome wire and DC power supply giving 3.2A and 32 Volt was passed.

 Step 3 - Using a thermometer for measuring, increase in temperature of the medium was noted and tabulated.

 Step 5 - The reading were compared with that of only nichrome wire

 Observation- The temperature of the water raised from 25 °C to 100 °C in 2 minutes but along with oil spluttering.

 Result- This method was ruled out.

×

## Sterilize media by indirect sterilization using powder ink

Date: 15th May 2019

 Time: 12:00 PM - 5:00 PM

 Total number of attendees: 4

 Number of male attendees: 2

 Number of female attendees: 2

 Ashtad, Shreya, Mihir, and Apeksha

 Aim- To determine the time required to raise the temperature of water from 25 °C to 100 °C by using ink powder particles (magnetic) by holding over induction coils.

 Protocol-

 Step 1 - Use 1gm of ink powder and mix it with 5 ml of water.

 Step 2 - Hold the tube over induction coils for heating.

 Step 3 - Using a thermometer inside the tube, increase in temperature is timed and tabulated.

 Observation- There was no effective rise in temperature

 Result - Method ruled out.

×

## Viscosity checking of medium that can be used

Date- 14-15 th June

Apeksha, Shreya

Aim- to check the time required for water, and various media to flow through Ostwalds viscometer

Protocol-

Step 1 - prepare 100 ml of water, nutrient broth, nutrient agar and lb broth

Step 2 - pass 10 ml of water through the viscometer and note time

Step 3 - pass 10 ml of nutrient broth and note time

Step 4 - pass 10 ml luria bertani broth through viscometer and note time

Step 5 - pass 10 ml nutrient agar through viscometer and note time

Observations-
Water passed through in 1 min 12 seconds
Nutrient broth passed through in 1 min 34 seconds
Luria Bertani broth passed through in 1 min 32 seconds
Nutrient agar passed through viscometer kept in a heated water bath ( 60-70°C in 1 hours 20 minutes

Result - Liquid media passed easily through the tiny capillary whereas heated agar was very difficult to pass. Hence tubing dimensions could be decided for further manufacture.

×

## Developing UV Chamber

Date: 17th - 23rd May 2019

Time: 12:00 PM - 5:00 PM

Total number of attendees: 4

Number of male attendees: 3

Number of female attendees: 1

 Ashtad, Sourabh, Mihir

 Aim- Circuit assembly inside an acrylic chamber with holding space for petri dishes in the centre. UV- C tubes installed inside the chamber on top and bottom to evenly irradiate the plates

×

## Sterilize using UV-C germicidal tube

Date: 6th- 11th June 2019

Time: 1:00 PM - 4:00 PM

Prerak, Chintan, Mihir, and Apeksha, Shreya

Aim- To determine effectivity of the manufactured UVC chamber by exposing plates with plain unsterilized medium to UV radiation for varied amounts of time and incubation of plates and enumeration.

Protocol-

Step 1 - Prepare nutrient media

Step 2 - After digestion, pour 20 ml of media onto plates and set to solidify.

Step 3 - Expose plates to UV by placing them into the chamber (while switched off!) and switching the chamber on after closing the chamber lids. The time of exposure was varied (2, 4, 5[Lid and no lid], 6, 8, 10).

Step 4 - Incubate plates at 37 °C for 24 hours.

Step 5 - Observe and look for contaminants.

Observation- There was a decrease in number of colonies as the time of exposure increased.

Results - 8 minutes of exposure to UV was enough for killing microbial population in media.

×

## Media preparation

Date- 1st July

Time - 9:00 am - 12:00 pm

Shreya, Apeksha

Aim- Preparation of 250 ml Sabourauds agar and 24 petri plates and set it for autoclaving

×

## Endophyte experiment

Date: 3rd July

Time: 9:00 am - 6:00 pm

Bhavna, Apeksha, Shreya, Chintan, Prerak

Aim - Isolation of endophytes from Neem (Azadirachta indica) and Tulsi (Ocimum tenuiflorum)

Protocol -

Step 1 - Prepare yeast media ( Sabouraud's agar).

Step 2 - alcohol sterilize the plant parts. Wash the plants, alcohol wash aseptically and wash with sterile water.

Step 3 - Cut the parts between burners ( cut small parts)

Step 4 - Using sterile spatula, transfer this to the agar plate.

Step 5 - Let the sap ooze out and incubate at 37 °C for 24- 48 hours.

Step 6 - After growth is observed, subculture the various colonies in sab broth. Centrifuge extract. Take supernatant and check antimicrobial activity against other organisms.

Step 7 - Co Culturing - bacteria and fungi. Measure again, using Buchner flask. Incubate for intervals of two hours and check OD. If OD decreases, it shows positive result, i.e. inhibition.

Observation- There was growth of colonies around the plant parts

Result- Presence of Endophytes have been observed and they have been screened for further use.

×

## Subculturing of endophytes to obtain a pure-culture

Apeksha- Subculturing of endophytes to obtain a pure-culture (✖5) with the assistance of Miss. Bhavna

×

## Replating of colonies

Date - 9th July 2019

Time- 12:00 pm - 3:00 pm

Apeksha, Shreya

Aim - Replating and segregation of colonies

Protocol -

Step 1 - Check if the plates show optimum growth

 Step 2 - Select colonies to further use in the process

Step 3 - pick these colonies and transfer them to fresh plates

 Step 4 - Incubate plates at Room temperature for 48 hours.

×

## Isolation of grown colonies

Date - 8th July 2019

Time - 12:00 pm - 4:00 pm

Apeksha, Shreya

Aim - Isolation of various different colonies found on sabourauds plates which had plant parts.
The colonies are differentiated and subcultured on sabourauds agar plates to obtain pure cuture.

Protocol-

Step 1 - Screen all part plates

Step 2 - Select and approve particular colonies with the desirable quality

Step 3 - pick these colonies

Step 4 - Take 20 ml of sterile sabourauds agar

Step 5 - Inoculate selected colonies into the agar and incubate at room temperature for 48 hours

Step 6 - Subculture the colonies into sabourauds agar slants and incubate at room temperature for 1 week.

Result - There were pure culture growth of colonies with no other colony presence.

×

## Checking growth

Date - 10 th July 2019

Time - 9:00 am

Shreya, Apeksha

Aim- Select colonies for mass growth.

Observation- Most plates showed pure culture characteristics. These colonies were chosen

×

## Mass culture

Date - 11th July to 18th July 2019

Shreya, Apeksha

Aim - Making large amounts of the chosen culture

Protocol-

Step 1 - Prepare sterile sabourauds broth 20 ml per tube (x 15)

Step 2 - Select desired colonies and inoculate into each tube

Step 3 - set on shaker at room temperature for 1 week

Step 4 - Check growth

×

## CAD modeling of U.V. chamber using AutoCAD Software

Date - 5th JULY

Ashtad- CAD modeling of U.V. chamber using AutoCAD Software

Sourabh + Mihir- U.V. Chamber assembled using laser cut black acrylic, circuits, hot-gun, and insulating tap

Apeksha + Prerak- SDG post on Instagram (#4)

Chintan- Studied the plants' sample requirements from the thesis- “Killer Activity of Yeast isolated from medicinal plants”

×

## New UV - C chamber manufacture

Date - 5 th July 2019

Mihir, Ashtad, Sourabh

Aim- Making a new furnished UVC Chamber

Protocol-

Step 1 - making acrylic sheets assembly by laser cutting

Step 2 - Refering to the CAD model made, the box is assembled

Step 3 - circuit connections

×

## Testing UVC chamber using plates

Date - 20th July - 21st July

Shreya, Apeksha, Chintan

Aim - to test microbicidal effect of the new UVC assembly by using plates

Protocol -

Step 1 - Make nutrient agar

 Step 2 - After digestion, nutrient agar is poured into the plates

Step 3 - the plates are exposed to the new chamber for 2, 4, 5, 6 ,8, 10 mins.

Step 4 - Incubation at room temperature for 24 hours

Observation - 5 minute plates showed maximum decrease in number

 Result - 5 minutes were enough to sterilize the media under this new chamber

×

## Spiral Tube experiments

Date - 7th JULY

Chintan + Mihir + Apeksha = Spiral Tube experiments

Apeksha + Shreya + Chintan = Bio-team meet-up over con-call

×

## Exploration of desired genes

Date - August

Shreya, Chintan, Apeksha

Aim - to select a protein/compound secreted by organism that has killing/ inhibitory activity on contaminating organisms.

 Notes-

Tyrosinase

Tyrosinase is an oxidase that is the rate -limiting enzyme for controlling the production of melanin. The enzyme is mainly involved in two distinct reactions of melanin synthesis; firstly, the hydroxylation of a monophenol and secondly, the conversion of an o-diphenol to the corresponding o-quinone. o-Quinone undergoes several reactions to eventually form melanin. Tyrosinase is a copper -containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation, as in the blackening of a peeled or sliced potato exposed to air. It is found inside melanosomes which are synthesised in the skin melanocytes. In humans, the tyrosinase enzyme is encoded by the TYR gene.

Clinical significance-

 A mutation in the tyrosinase gene resulting in impaired tyrosinase production leads to type I oculocutaneous albinism, a hereditary disorder that affects one in every 20,000 people.
Tyrosinase activity is very important. If uncontrolled during the synthesis of melanin, it results in increased melanin synthesis. Decreasing tyrosinase activity has been targeted for the betterment or prevention of conditions related to hyperpigmentation of the skin, such as melasma and age spots.
Several polyphenols, including flavonoids or stilbenoid, substrate analogues, free radical scavengers, and copper chelators, have been known to inhibit tyrosinase. Henceforth, the medical and cosmetic industries are focusing research on tyrosinase inhibitors to treat skin disorders.

Reverse Translate results

Results for 625 residue sequence "BAC87843.1 tyrosinase precursor 1 [Illex argentinus]" starting "MESYRLLVLV"

 >reverse translation of BAC87843.1 tyrosinase precursor 1 [Illex argentinus] to a 1875 base sequence of most likely codons.

atggaaagctatcgcctgctggtgctggtgagcgcgtttggcctgtgccaggcgatggtg
gatgtgagccagagcgatggcctgcagagctgcctggatcgctttgcggatgataccagc
acctttagccagcaggaacagctgagcctgtgcagcaaatattatatgcagaaaaactgg
aaaagcgcggatgtgagcaaaccgaaaattagcaccctggcgaccatgagcccgcaggaa
tatattcagagcctgattgatcgctttaccgcggaagcgcgcaacccgcagggccgccgc
gtgcgcaaagaatatcgcatgatgaccaacgaagaacgcgataactatcatcgcgcgatt
gtgatgctgaaacaggataccaccgtgctgccgaacaaatttgaaattattgcggatctg
catgcgggcagcgtgaccaacagcgcgcatggcggcccgggctttctgccgtggcatcgc
atttatatgatgatttgggaagaaggcctgcgcgaacaggtgccgaccgtggtggtgccg
tattgggatgtgacccgcgatagcgcgatggatgatccgcgccgcagcattgtgtggagc
ccgcagtttcagggcaacggccatggcctggtgaccgtgggcccgtttgcggattggacc
accggctatggcccgctgcatcgcaactatgcggtgtttacccatctgctgacccgcgcg
aacattcagaccgtgtttaccgaacgcaccattgcggaaattagccagctgaccgcgaac
gatcagcgctatgtgtttgaactgtatcataacaacattcatgattggattggcggcacc
gtgagcgtgcaggcgtgggcgagctttgatccggcgtttatgctgattcatggctatgtg
gattatatttggtatcgctttcaggaaatgcagctggaactgggcggcattgatattagc
gtggattatccgtttaccgcgaaccatcagattctgaacggcaccgcgtttgatggcgaa
gaaccggtgggcctgattccgggcatgaccaaccgcgaagcggtggcggaaggcgtggtg
tatatgaacctgatgaactatgaagaagcgcagagcgattgcgatcagcagaccccgtgc
ccgccgaactatgaatgcgtggatggcttttgcgcgagccgcgcggtggataacgatgtg
tgcaaccagattcagccgctgcagaacaacttttgcattaacaaagaatgcgatgtgagc
ctgtttagctttctggcggtggaaattattcatgaacgcatggaaaacctgtgcaacatg
ggcaactttccggtgcgccagtggctggcggataaaaccgcggatatttatcgcgaaagc
gcgagcgtgattcatcagggcaaatatagcaacagcctgagcgatatgtgcggccgcccg
ggcggctgctgcaaaccggtggaacgcgtgaacattcaggtgagcggcaacgatggcgat
ctgtggctgtatcgcgaaagcgcgtttgtggatgcgcgcctggcggtgagccatagccag
atgtttgtggcggtgcgccgcgtgccgattggccgctttctgatttttgcggcggatgaa
tatggcaacctgtgcgatgcgtatctggtggatacctttggcaacaaaattctgctgcgc
cgcagcgaaggcattattattagcgaagatgatccgcgcctgagcaacaccctggcggaa
gcggaaagcaaaatgtttgattatcagaacggccaggatctgccgccgaacgtgctgcag
aaccagtatgtgctgagctttcattgccgcgcggatcgcaacctgggcccgagcgtgcgc
Aacggcaacaaaaaa

 >Standard RBS

>BBa\_B0034 Part-only sequence (12 bp)

aaagaggagaaa

(TIPS- dont use T7 promoter as it is derived from virus find others)

>CODON OPTIMISED AND RFC{10} compatibale Tyrosinase precursor
1 ATGGAATCTT ATCGCCTGCT GGTGCTGGTT TCTGCTTTTG GCCTGTGCCA GGCCATGGTT GATGTTTCTC
71 AGAGCGACGG CCTGCAAAGC TGTCTGGATC GCTTCGCTGA CGATACGAGC ACGTTTAGCC AGCAAGAACA
141 GCTGAGCCTG TGTTCCAAAT ACTATATGCA GAAAAATTGG AAATCTGCGG ATGTTAGCAA ACCGAAAATC
211 TCTACCCTGG CGACTATGAG CCCGCAGGAG TACATTCAGA GCCTGATCGA TCGTTTCACC GCAGAAGCAC
281 GTAACCCGCA GGGTCGTCGC GTTCGTAAAG AGTACCGCAT GATGACCAAT GAAGAACGCG ATAACTACCA
351 CCGTGCGATC GTGATGCTGA AACAGGACAC TACGGTGCTG CCGAACAAGT TCGAGATCAT CGCAGACCTG
421 CATGCTGGTT CCGTAACCAA CAGCGCACAT GGTGGTCCAG GCTTTCTGCC GTGGCATCGC ATTTACATGA
491 TGATCTGGGA AGAAGGCCTG CGCGAGCAAG TTCCGACTGT TGTAGTCCCT TATTGGGATG TCACCCGCGA
561 TTCCGCTATG GACGATCCTC GTCGCAGCAT CGTTTGGAGC CCACAGTTTC AGGGCAACGG CCACGGCCTG
631 GTTACCGTTG GTCCGTTCGC TGACTGGACG ACCGGTTACG GTCCTCTGCA CCGTAACTAT GCCGTTTTCA
701 CCCACCTGCT GACCCGTGCG AACATCCAGA CTGTTTTCAC CGAACGTACT ATCGCAGAGA TCAGCCAACT
771 GACCGCGAAC GATCAGCGTT ATGTATTTGA GCTGTACCAC AACAATATCC ACGATTGGAT TGGTGGTACT
841 GTATCCGTCC AGGCATGGGC AAGCTTCGAT CCGGCTTTCA TGCTGATTCA CGGTTATGTG GACTACATCT
911 GGTACCGCTT CCAGGAAATG CAGCTGGAAC TGGGTGGTAT TGACATTAGC GTTGACTACC CGTTCACCGC
981 GAACCACCAG ATCCTGAACG GTACCGCATT TGATGGTGAG GAACCGGTAG GCCTGATCCC TGGCATGACC
1051 AACCGTGAAG CAGTGGCGGA AGGCGTGGTA TACATGAACC TGATGAATTA CGAAGAAGCA CAGTCCGACT
1121 GTGATCAGCA GACCCCGTGT CCGCCGAACT ACGAATGTGT TGATGGTTTT TGCGCATCTC GTGCAGTAGA
1191 TAACGACGTT TGCAACCAGA TCCAGCCTCT GCAAAACAAC TTCTGCATTA ACAAAGAATG CGATGTTAGC
1261 CTGTTCAGCT TCCTGGCGGT CGAAATCATC CACGAACGCA TGGAAAACCT GTGTAATATG GGCAACTTCC
1331 CGGTACGTCA GTGGCTGGCG GATAAAACGG CGGACATTTA CCGTGAATCC GCTTCTGTGA TCCATCAGGG
1401 TAAATACAGC AACTCTCTGT CTGATATGTG TGGCCGTCCA GGTGGCTGCT GCAAACCGGT TGAACGTGTC
1471 AATATCCAGG TATCCGGCAA TGATGGTGAC CTGTGGCTGT ACCGCGAAAG CGCTTTCGTT GACGCCCGTC
1541 TGGCTGTATC CCATAGCCAG ATGTTCGTTG CGGTACGTCG CGTGCCGATT GGTCGTTTCC TGATCTTCGC
1611 AGCAGACGAA TACGGCAACC TGTGCGATGC ATACCTGGTT GATACTTTTG GTAACAAAAT TCTGCTGCGT
1681 CGCTCCGAAG GCATCATCAT TTCTGAAGAT GACCCGCGTC TGTCTAACAC CCTGGCGGAA GCAGAAAGCA
1751 AGATGTTCGA CTATCAGAAT GGTCAAGATC TGCCACCAAA CGTTCTGCAA AACCAATATG TTCTGTCCTT
1821 CCACTGCCGT GCAGACCGCA ACCTGGGTCC ATCTGTTCGT AACGGTAACA AAAAA

>reverse translation of BAC87843.1 tyrosinase precursor 1 [Illex argentinus] to a 1875 base sequence of consensus codons.
atggarwsntaymgnytnytngtnytngtnwsngcnttyggnytntgycargcnatggtn
gaygtnwsncarwsngayggnytncarwsntgyytngaymgnttygcngaygayacnwsn
acnttywsncarcargarcarytnwsnytntgywsnaartaytayatgcaraaraaytgg
aarwsngcngaygtnwsnaarccnaarathwsnacnytngcnacnatgwsnccncargar
tayathcarwsnytnathgaymgnttyacngcngargcnmgnaayccncarggnmgnmgn
gtnmgnaargartaymgnatgatgacnaaygargarmgngayaaytaycaymgngcnath
gtnatgytnaarcargayacnacngtnytnccnaayaarttygarathathgcngayytn
caygcnggnwsngtnacnaaywsngcncayggnggnccnggnttyytnccntggcaymgn
athtayatgatgathtgggargarggnytnmgngarcargtnccnacngtngtngtnccn
taytgggaygtnacnmgngaywsngcnatggaygayccnmgnmgnwsnathgtntggwsn
ccncarttycarggnaayggncayggnytngtnacngtnggnccnttygcngaytggacn
acnggntayggnccnytncaymgnaaytaygcngtnttyacncayytnytnacnmgngcn
aayathcaracngtnttyacngarmgnacnathgcngarathwsncarytnacngcnaay
gaycarmgntaygtnttygarytntaycayaayaayathcaygaytggathggnggnacn
gtnwsngtncargcntgggcnwsnttygayccngcnttyatgytnathcayggntaygtn
gaytayathtggtaymgnttycargaratgcarytngarytnggnggnathgayathwsn
gtngaytayccnttyacngcnaaycaycarathytnaayggnacngcnttygayggngar
garccngtnggnytnathccnggnatgacnaaymgngargcngtngcngarggngtngtn
tayatgaayytnatgaaytaygargargcncarwsngaytgygaycarcaracnccntgy
ccnccnaaytaygartgygtngayggnttytgygcnwsnmgngcngtngayaaygaygtn
tgyaaycarathcarccnytncaraayaayttytgyathaayaargartgygaygtnwsn
ytnttywsnttyytngcngtngarathathcaygarmgnatggaraayytntgyaayatg
ggnaayttyccngtnmgncartggytngcngayaaracngcngayathtaymgngarwsn
gcnwsngtnathcaycarggnaartaywsnaaywsnytnwsngayatgtgyggnmgnccn
ggnggntgytgyaarccngtngarmgngtnaayathcargtnwsnggnaaygayggngay
ytntggytntaymgngarwsngcnttygtngaygcnmgnytngcngtnwsncaywsncar
atgttygtngcngtnmgnmgngtnccnathggnmgnttyytnathttygcngcngaygar
tayggnaayytntgygaygcntayytngtngayacnttyggnaayaarathytnytnmgn
mgnwsngarggnathathathwsngargaygayccnmgnytnwsnaayacnytngcngar
gcngarwsnaaratgttygaytaycaraayggncargayytnccnccnaaygtnytncar
aaycartaygtnytnwsnttycaytgymgngcngaymgnaayytnggnccnwsngtnmgn
Aayggnaayaaraar

Cephalopod ink contains a number of chemicals in a variety of different concentrations, depending on the species. However, its main constituents are melanin and mucus. It can also contain, among other things, tyrosinase, dopamine and L-DOPA, as well as small amounts of free amino acids, including taurine, aspartic acid, glutamic acid, alanine and glycine. Cephalopod ink has, as its name suggests, been used in the past as ink for pens and quills; the Greek name for cuttlefish, and the taxonomic name of a cuttlefish genus, Sepia, is associated with the brown colour of cuttlefish ink (for more information, see sepia).
Modern use of cephalopod ink is generally limited to cooking, primarily in Japan and the Meditteranean , where it is used as a food coloring and flavouring, for example in pasta and sauces. For this purpose it is generally obtainable from fishmongers, gourmet food suppliers, and is widely available in markets in Japan. The ink is extracted from the ink sacs during preparation of the dead cephalopod, usually cuttlefish, and therefore contains no mucus.
Studies have shown that cephalopod ink is toxic to some cells, including tumour cells. It is being researched in mice for its antitumor activity against Meth-A fibrosarcoma. It currently remains unclear however if any of the antitumor activity of squid ink can be obtained from oral consumption, and this is indicated as an area for future investigation.
Tyrosinase activity is very important. If uncontrolled during the synthesis of melanin, it results in increased melanin synthesis. Decreasing tyrosinase activity has been targeted for the betterment or prevention of conditions related to hyperpigmentation of the skin, such as melasma and age spots.
Antimicrobial activity of tyrosinase The antimicrobial activity of tyrosinase was assayed against Salmonella typhimurium [ATCC 14028], Staphylococcus aureus subsp. aureus[ATCC 25923], Staphlococcus aureus [MRSA] [ATCC 43300], Bacillus cereus [ATCC 33018], Bacillus subtilis subsp spizizenii [ATCC 6633], Listeriamonocyogenes [ATCC 7644], Listeria innocua [ATCC 33090], Escherichia coli [ATCC 11775], Pseudomonas aerugmosa [ATCC 10145] Candida albicans [ATCC 26555] and Aspergillus niger [nrrl 326] [19]. The susceptibility was assayed using CLCI, M44A on Mueller Hinton agar plate.Out of nine tested pathogenic bacterial species, the enzyme could inhibit the growth of four species only, Bacillus subtilis, Staphylococcus aureus subsp. aureus, Pseudomonas aeruginosa and Escherichia coli, with inhibition zone diameters of 9, 7, 6 and 6 mm, respectively [Table 9]. Tyrosinase showed no antifungal activity either on the filamentous fungal species Aspergillus niger or on the unicellular fungal species Candida albicans. No available data could be obtained in that field except that of Nosanchuk and Casaevell [63] who reported an antimicrobial action of tyrosinase which contributed to microbial pathogenesis.

Sepia pharaonis

Common cuttlefish

>tr|Q7YT36|Q7YT36\_SEPOF Tyrosinase OS=Sepia officinalis OX=6610 PE=2 SV=1
MSLFGICKAMVNISQSNMLQNCFDRFASDSSILTEQEQVSLCFKYYTQNNLQSADVPNSK
VSPLATMTPEEYIQSLIGRFTAEARNPQNKRVHKEYRMMTNEERENYHQAIIMLKQDTTV
LPNKFEVIADLHVGFITNSAHGGPGFLPWHRIYMMIWEEGLREQIPSVVVPYWDVTRDSA
LEDPRRSIIWSPEFQGNGHGLVTSGPFAGWLTGYGPLHRNYAVFTHLLTRENVRTVFTQK
SLAQISQLTANEQRYIFELYHNNIHDWIGGTVSVQAWASFDPAFMLIHGYVDYIWYRFQE
MQLEANINISEDYPMTSNHQILNGTAFDADAPIGIIAGMSNREAVAESVVYMNLIDYEEA
PTDCDEETPCGSPYYECVNGFCASRIIANDVCSQTIPLQNNFCVDKICDTGLFSFLPVEV
IHERLESLCDMSNFPVRQWVPDNTMDIYRESANIVHQDRYFNRPSDMCGRPGGCCKPVER
VNIQVNGNDGNLWLYKESVYVDTRLAVSHSHMFVAIKRAPIGRFLIFAADEYGNLCDTYL
LDTFGNRILLRRNEGIIISENDQRISSTLAEAELKMFNYVTGQSLPTVRQDQYVISFHCR
ADRNQN

Aspergillus oryzae (strain ATCC 42149 / RIB 40) (Yellow koji mold)

>sp|Q00234|TYRO\_ASPOR Tyrosinase OS=Aspergillus oryzae (strain ATCC 42149 / RIB 40) OX=510516 GN=melO PE=1 SV=1 MASVEPIKTFEIRQKGPVETKAERKSIRDLNEEELDKLIEAWRWIQDPARTGEDSFFYLA GLHGEPFRGAGYNNSHWWGGYCHHGNILFPTWHRAYLMAVEKALRKACPDVSLPYWDESD DETAKKGIPLIFTQKEYKGKPNPLYSYTFSERIVDRLAKFPDADYSKPQGYKTCRYPYSG LCGQDDIAIAQQHNNFLDANFNQEQITGLLNSNVTSWLNLGQFTDIEGKQVKADTRWKIR QCLLTEEYTVFSNTTSAQRWNDEQFHPLESGGKETEAKATSLAVPLESPHNDMHLAIGGV QIPGFNVDQYAGANGDMGENDTASFDPIFYFHHCFIDYLFWTWQTMHKKTDASQITILPE YPGTNSVDSQGPTPGISGNTWLTLDTPLDPFRENGDKVTSNKLLTLKDLPYTYKAPTSGT GSVFNDVPRLNYPLSPPILRVSGINRASIAGSFALAISQTDHTGKAQVKGIESVLSRWHV QGCANCQTHLSTTAFVPLFELNEDDAKRKHANNELAVHLHTRGNPGGQRVRNVTVGTMR

Solenopsis invicta (Red imported fire ant) (Solenopsis wagneri)

>sp|Q8WP90|PBGP9\_SOLIN Pheromone-binding protein Gp-9 OS=Solenopsis invicta OX=13686 GN=Gp-9 PE=1 SV=1
MKTFVLHIFIFALVAFASASRDSARKIGSQYDNYATCLAEHSLTEDDIFSIGEVSSGQHK
TNHEDTELHKNGCVMQCLLEKDGLMSGADYDEEKMREDYIKETGAQPGDQRIEALNACMQ
ETKDMEDKCDKSLLLVACVLAAEAVLADSNEGA

Solenopsis invicta virus 3 (SINV-3)

 >sp|C1JCT1|POL\_SINV3 Polyprotein OS=Solenopsis invicta virus 3 OX=631345 PE=1 SV=1
MSEKTQTFVQNETHVLDMTSDFKSDLSLEKVTSSVEQTDDLVSKIINNNDLDIKDLSFLR
NLLLSTLQYLGIAKFVAINITLSILSILMLLINSCAKFTRIVNLSSHILNIITTLGLYFQ
VSSMEIEEITQTFENEFGTYDDDKILSHYIKICNLPNRKDVYEYISLNDLKYKIKLPDIS
FYELKNDILSKNKNLHLWIFQKFTDEFLAMWFGVQPYRISNLREMLVISRQGFIPKDLFN
EIRKLCNMGVSVIISFIQSKLFDEPFKKRDCTQALKDASVISSPFDTLWNLISKQVCDNS
AEERFTQTILDFTSEFDNFLGIPNYKFAKNQKLVNTISKSLDACAKFIRDCPKDKQTEIF
PLQGLHTATVKRRNEILTNVMPKFARQEPFVVLFQGPGGIGKTHLVQQLATKCVNSFYQD
HEDDYIEISPDDKYWPPLSGQRVAFFDEAGNLNDLTEDLLFRNIKSICSPAYFNCAAADI
EHKISPCPFELVFATVNTDLDTLQSKISSTFGQASVFPIWRRCIVVECSWNEKELGPFNY
KNPSGHRSDYSHITMNYMSYDDKTQKLALEKEINFDTLFDMIRLRFRKKQQEHDTKISIL
NNEIQRQSNSKQHFSVCLYGEPGQGKTYNLNKLITTFANATNLKIGSEEKPSIHIFDDYI
KDENDENCSKFMDIYNNKLPNNSVIFSATNVYPKTHFFPTFFLTNLIYAFIQPFKQVGLY
RRLGFDGYTDIPNSSVNAPIFVQNFKFYERKQHICYFLSLEFLKNIICYIFFFLYFPLKF
IKKIDLIEIKDVNKYVYDRYINFLSLSKQIEIVEYPPNLENVEFDFRFNMNKFHRVSFNN
PFELDKYIHFNKNSYENLLHFDWKMYLSPRVKHRLALSYEKFFITISEVNKEIIIEELKR
YVLLFKQFNIDPNMEINLGEYGSFYYINGKIHLMTINIESNVSEIPVFTDGDYVYISEHK
IPVIDLFDNININSKYNLSFDQSIALNSFKTGDSFYSNAKVRKSLSKFVLLNYQTKFKLY
LKEAKDKVKNFIETPIGHLLSILLTIFVICYASFKIYSKFSNFFSKDQAIEDQRKGEKKI
KKITNYDSDGVQPQRKGEKKIKKVTNYDSDGVQPQSNVKVEEEIKLVFDPTGQKLLFGND
FTSELETLVELEKDDEEFTKSKIDNKSMAGLRREVRRRRYARSKKAQIEKQEVLTLPDVN
GFEGGKPYFQIAEEKARKNLCQIYMIANNENCIASKFSDHIVCYGLFVFKKRLASVGHIV
EALKCAPGYNLYAGCDQFNGKLYKMNLVRNYRKRELSVWDVDCPNDFVDLTSFFIPKEEL
YDAENCNTVLGRFGMNKREVYLYGNCEFIQEFFKVDNKGAQEFGYIDWATVDITLTTGGD
CGLPYYICERKKFHNKIMGLHFAGNNVNHKTIGMSALIYKEDLVVWKGAERQSKCKFCDV
KDIIIAQPDIPKEKYKGYNHEIVWNSLHESSPTTLNEELEHYLNIFPKFTGTIIKHSGDK
FYGSVKHSHTQFISKFKTELTVTNGWKLSTAGDCQFESNHISPNTEVMYRVVDVQFNSIF
KAFKSQPYIKNFRLIANVYEKDGKQRVTILTIIPVSDFNVKQQTVRQALVPLHLNEDEEV
YVTEDVSDIFKTAIKRKQRGILPDVPYETVENETVEILGITHRNMTPEPAQMYKPTPFYK
LALKFNLDHKLPVNFNMKDCPQEQKDMMVLDRLGQPNPRITQSLKWAHKDYSPDYELRKY
VKEQYMCNIMEYYAGCNLLTEEQILKGYGPNHRLYGALGGMEIDSSIGWTMKELYRVTKK
SDVINLDSNGNYSFLNNEAAQYTQELLKISMEQAHNGQRYYTAFNELMKMEKLKPSKNFI
PRTFTAQDLNGVLMERWILGEFTARALAWDENCAVGCNPYATFHKFATKFFKFKNFFSCD
YKNFDRTIPKCVFEDFRDMLIQANPHMKNEIYACFQTIIDRIQVSGNSILLVHGGMPSGC
VPTAPLNSKVNDIMIYTAYVNILRRADRGDITSYRYYRDLVCRLFYGDDVIIAVDDSIAD
IFNCQTLSEEMKILFGMNMTDGSKSDIIPKFETIETLSFISRFFRPLKHQENFIVGALKK
ISIQTHFYYATDDTPEHFGQVFKTIQEEAALWEEEYFNKIQSYIQEIIRKFPEISKFFNF
ESYKSIQKRYIMNGWNEFVKLEKLDLNLNKKKSSKVTGIHSKQYSKFLKFLSRIENEKAA
LEGNFNKESVNTWYFKMSKAMHLNEIFQKGLISKPLAEFYFNEGQKMWDCNITFRRSKDD
LPFTFSGSGTTKACAREQAAEEALVLFSQEDEIVRQINDIQSDCKFCKKMIRYKKLLSGV
SIQRQMNVSKITENHVPSAGMMATDPSVAPDSGIATNTQTPSISRVLNPIARALDNPAGT
GAPFDKHTYVYNVFTRWPEMSTVVNKSLAAGAEVFKISLDPNKLPKRILQYIQFHKTIIP
QIEVQILIGGAAGTVGWLKVGWVPDASTAKKYSLDDLQLVASETINLNSTITMSMIINDS
RRNGMFRLTKSDPEPWPGIVCLVEHPITNVQRNDDVNYPVIVSVRLGPDCQLMQPYNDLN
Aestero saponins

>sp|P84117|1-51
FNKLKQGSSKRTCAKCFRKIMPSVHELDERRRGANRWAAGFRKCVSSICRY

Hymenoptaecin

>sp|Q10416|HYTA\_APIME Hymenoptaecin OS=Apis mellifera OX=7460 PE=2 SV=1
MKFIVLVLFCAVAYVSAQAELEPEDTMDYIPTRFRRQERGSIVIQGTKEGKSRPSLDIDY
KQRVYDKNGMTGDAYGGLNIRPGQPSRQHAGFEFGKEYKNGFIKGQSEVQRGPGGRLSPY
FGINGGFRF

Reverse transciption

Reverse Translate results

Results for 129 residue sequence "sp|Q10416|HYTA\_APIME Hymenoptaecin OS=Apis mellifera OX=7460 PE=2 SV=1" starting "MKFIVLVLFC"

>reverse translation of sp|Q10416|HYTA\_APIME Hymenoptaecin OS=Apis mellifera OX=7460 PE=2 SV=1 to a 387 base sequence of most likely codons.
atgaaatttattgtgctggtgctgttttgcgcggtggcgtatgtgagcgcgcaggcggaa
ctggaaccggaagataccatggattatattccgacccgctttcgccgccaggaacgcggc
agcattgtgattcagggcaccaaagaaggcaaaagccgcccgagcctggatattgattat
aaacagcgcgtgtatgataaaaacggcatgaccggcgatgcgtatggcggcctgaacatt
cgcccgggccagccgagccgccagcatgcgggctttgaatttggcaaagaatataaaaac
ggctttattaaaggccagagcgaagtgcagcgcggcccgggcggccgcctgagcccgtat
tttggcattaacggcggctttcgcttt
>reverse translation of sp|Q10416|HYTA\_APIME Hymenoptaecin OS=Apis mellifera OX=7460 PE=2 SV=1 to a 387 base sequence of consensus codons.
atgaarttyathgtnytngtnytnttytgygcngtngcntaygtnwsngcncargcngar
ytngarccngargayacnatggaytayathccnacnmgnttymgnmgncargarmgnggn
wsnathgtnathcarggnacnaargarggnaarwsnmgnccnwsnytngayathgaytay
aarcarmgngtntaygayaaraayggnatgacnggngaygcntayggnggnytnaayath
mgnccnggncarccnwsnmgncarcaygcnggnttygarttyggnaargartayaaraay
ggnttyathaarggncarwsngargtncarmgnggnccnggnggnmgnytnwsnccntay
Ttyggnathaayggnggnttymgntty

×

## Model testing using various media

Date- 15th September

Apeksha

Aim- To see the effect of UV on organisms growing on different media.

Protocol-

Step 1 - Prepare Luria Bertani agar, nutrient agar, MacConkey agar, Mueller Hinton agar, Sabourauds agar

Step 2 - Pour non- sterile media into sterile plates aseptically.

Step 3 - Expose plates to UV in the UVC chamber for 5 minutes

Step 4 - Incubate at room temperature for 24 hours.

Step 5 - Observe

Result - MacConkey media and LB media showed minimum growth of organisms and Mueller Hinton agar showed very scanty growth of contaminants.Maximum growth was found in Nutrient agar.

×

## Prototype building

Aim - To build a prototype of our experiment using CAD model
Ashtad

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Bioriidl Somaiya/Results

Project

![](https://static.igem.org/mediawiki/2019/1/1b/T--Bioriidl_Somaiya--ham.png)
[![igem logo](https://static.igem.org/mediawiki/2019/d/de/T--Bioriidl_Somaiya--igem.png)](https://2019.igem.org/Team%3ABioriidl_Somaiya)

* Team
  + Team Members
  + Collaboration
* Project
  + Description
  + Design
  + Experiments
  + Notebook
  + Contributions
  + Results
  + Demonstration
  + Improvements
* Parts
  + Parts Overview
  + Basic Parts
  + Composite Parts
  + Parts Collection
* Safety
* Human Practices
  + Human Practices
  + Education and Engagement
* Awards
  + Entrepreneurship
  + Hardware
  + Software
* Judging Form

# Ideation

Our journey began in January of 2019, when the iGEM meetings began.
People from a number of fields aggregated to come and speak about
their ideas and prototypes of the experiment to be carried out. The
idea kept coming aplenty but the decision of choosing the best was
the hardest one ever!

![](https://static.igem.org/mediawiki/2019/0/03/T--Bioriidl_Somaiya--ideation.jpg)

* But after so much of the discussion and plotting, we finalized on
  going forward with a project which respected our fundamentals and
  would improve the lives and time of many. We needed to now focus on
  the most primary level in almost all our experiments. The autoclave.
  It is certain that autoclave usage is time consuming and very
  taxing. It needs inspection, tests and along with this you also have
  to wait for the media plates to dry! To battle this we figured out a
  solution, after tons and tons of experimentation.

# The Solution

Media generally used in labs is the nutrient , MacConkey, LB,
Sabouraud's media ( broth and agar). For effective and quick
sterilization of the desired media, the following conditions were
tested and proved - The heating of the media! The most crucial part
of our project. Media needs to be heated to high temperatures of
approximately 105°C to achieve killing of bacteria, spores and
fungi. For getting this, we used our nichrome wire, wound around a
basic dilution tube containing media or water which is later tested
using a thermometer for checking rate of increase in temperature. We
found that the media heated up in approximately 1 - 2 minutes, which
was a very good result, compared to the 2.5 hours wasted normally in
an autoclave. For preservation of heat and consistent spread, we
used the oil surrounds which were not as efficient as expected. The
amount of reduction of cells were seen when we used yeast cells for
control and heating this way showed a huge decrease in cell count

![](https://static.igem.org/mediawiki/2019/2/28/T--Bioriidl_Somaiya--res1.jpg)

UV C is known for it microbicidal properties, which were employed
here to kill any remaining bacteria or contaminants. The media is
passed through the heating coils at high temperatures and then held
in the UV chamber with the UV tube for approximately 5 minutes,
which reduces count of bacteria further ( UVC causes dimerisation of
thymines, the accumulation of which prevents the binding of
polymerase due to which replication is inhibited and hence the cell
is led to death)

![](https://static.igem.org/mediawiki/2019/e/e6/T--Bioriidl_Somaiya--res2.jpg)

These parameters would handle most of the contaminant non-
specifically leading to reduction of count of cells in the media.
For the biological part, we targeted Nisin A, for which we had
reference of proof from an inhouse researcher. We obtained our Nisin
from him and added 2ml of 2% Nisin to prewarmed 10 ml of media. The
media was left to activate for 10 minutes and then the liquid was
passed through the sterilization unit ( heater plus UV chamber). The
results showed completely sterile medium. Since the enzymes are
harmful for organisms, we needed to test the final medium for
viability for growth of further organisms. The enzyme is heated in
the sterilization unit and hence is degraded due to protein
denaturation. This was tested by growing of other organisms on the
final media. The results showed healthy, normal growth of bacteria.
The gene hence selected was that of Nisin A, Subtilosin-A and
Sporulation Killing Factor (SKF). Subtilosin- A and SKF were tested
similarly and obtained from inhouse researchers. Testing prototype-
The final model shows entry of media mix ( non sterile media plus
enzyme mix) through the entry cone and its passage through the
heating coils ( nichrome wire complex) for a duration of 2 minutes
leading to finally, the UV chamber for holding ( 6 minutes) and
sterilization.

![](https://static.igem.org/mediawiki/2019/7/70/T--Bioriidl_Somaiya--res3.jpg)

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