Team:Bioriidl Somaiya/Experiments

Experiments

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.

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.

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.

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.

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.

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.

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.

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.

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.

Experiment No. 10

Media preparation

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

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.

Experiment No. 12

Subculturing of endophytes to obtain a pure-culture

Experiment No. 13

Replating of colonies

  • Aim: Replating and segregation of colonies.

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.

Experiment No. 15

Checking growth

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

Experiment No. 16

Mass culture

  • Aim: Making large amounts of the chosen culture.

Experiment No. 17

CAD modeling of U.V. chamber using AutoCAD Software

Experiment No. 18

New UV - C chamber manufacture

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.

Experiment No. 20

Spiral Tube experiments

Experiment No. 21

Exploration of desired genes

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

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.

Experiment No. 23

Prototype building

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

Bioriidl © Copyright 2019. All rights reserved.

×

Testing the efficiency of nichrome wire


Date: 6th April 2019
Time: 2:00 PM - 4:00 PM
Total number of attendees: 8
Number of male attendees: 5
Number of female attendees: 3
Shreya, Prerak, Chintan, Sourabh, Ashtad, Mihir and Apeksha

Aim - To check whether the current passed through nichrome wire increase the temperature.

Protocol -
Step 1: - Nichrome wire is twisted around the mercury bulb of the thermometer.
Step 2: - Two sprung metal clips were attached on either end 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 average result

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.

×

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

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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.

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Prototype building

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