iGEM 2019 — Team MITADTBIO_Pune

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# Team:MITADTBIO Pune/Notebook

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  * **PROJECT▾**
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    + [**| MATHEMATICAL MODELLING**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Model)
    + [**|FUTURE PROSPECTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/3D%20MODEL)
  * [**PARTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Basic_Part)
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  * **COMMUNITY▾**
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    + [**| SURVEY**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Survey)
  * **AWARDS▾**
    + [**| MEDALS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Medal)

# 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:MITADTBIO Pune/Experiments

* [**HOME**](https://2019.igem.org/Team%3AMITADTBIO_Pune)* **TEAM▾**
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  * **PROJECT▾**
    + [**| DESCRIPTION**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Description)
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    + [**| EXPERIMENTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Experiments)
    + [**| RESULTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Results)
    + [**| MATHEMATICAL MODELLING**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Model)
    + [**|FUTURE PROSPECTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/3D%20MODEL)
  * [**PARTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Basic_Part)
  * [**SAFETY**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Safety)
  * **COMMUNITY▾**
    + [**| HUMAN PRACTICES**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Human_Practices)
    + [**| PUBLIC ENGAGEMENT**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Public_Engagement)
    + [**| SURVEY**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Survey)
  * **AWARDS▾**
    + [**| MEDALS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Medal)

![](https://static.igem.org/mediawiki/2019/3/35/T--MITADTBIO_Pune--expheader.png)

##### **"Try and try until your PCR starts working" ~ your average iGEMmer**

###### Standardizing protocols was a crazy and time consuming journey for us. We therefore present to you firstly with a list of all the approaches we tried for transformation and making compentent cells, protocols for the procedures that did work for us, and assays we carried out for our project.

## **APPROACHES**

Competent cells

###### | METHOD 1 | METHOD 2 | METHOD 3 | METHOD 4 | | --- | --- | --- | --- | | 1] 1ml of O/N DH5a culture in fresh 50 ml LB for 3 hours. Keep on ice for 30 min | 1]1ml of O/N DH5a culture in fresh 50 ml SOB for 3 hours | JM109 Competent Cells from Promega | 1]1ml of O/N DH5a culture in fresh 50 ml LB for 3 hours | | 2]Centrifuge 25 ml for 10 min at 4000rpm | 2]Centrifuge 25 ml for 10 min at 3000rpm | | 2]Centrifuge 25 ml for 10 min at 6000rpm | | 3]Add 200ul MgCl2 of 100 mM to pellet. Ice for 30 min | 3]Re-suspend in 6ml of CCMB80( tip : add 3ml first to resuspend the cells. When cells are in suspension, add another 3ml CCMB80 buffer for a total of 6ml ). Ice for 20 min | | 3]Resuspend in 6.4ml of HTB | | 4]Centrifuged for 10 min at 4000rpm | 4]Centrifuged for 10 min at 3000rpm | | 4]Centrifuged for 15 minutes at 3500rpm | | 5]Add 100ul CaCl2 of 100 mM to pellet. Ice for 20 min | 5]Re-suspend in 4ml of CCMB80 (tip : add 2ml first to resuspend the cells. When cells are in suspension, add another 2ml CCMB80 buffer for a total of 4ml) . Ice for 20 min | | 5]Re-suspend in 1.6 ml of HTB. Add 120uL DMSO | | 6]Make aliquots of 50µl | 6]Make aliqouts of 50 ul. | | 6]Make aliqouts of 200ul. | Preparation of medium 1]SOC Broth/agar | Mix | Quantity(100ml) | | --- | --- | | 0.5%yeast extract | 0.5g | | 2% tryptone | 2g | | 10mM NaCl | 0.2ml | | 2.5mM KCl | 0.5ml | | 20mM MgSO4 | 2ml | | 10mM MgCl2 | 2ml | | 20mM glucose | 2ml | | Agar | 1.5g | 2]SOB Broth/agar | Mix | Quantity(100ml) | | --- | --- | | 0.5%yeast extract | 0.5g | | 2% tryptone | 2g | | 10mM NaCl | 0.2ml | | 2.5mM KCl | 0.5ml | | 20mM MgSO4 | 4ml | | Agar | 1.5g | 3]Luria Broth/agar | Mix | Quantity(100ml) | | --- | --- | | Luria Broth | 2.0g | | Luria agar | 3.5g | 4]CCMB80 buffer (pH 6.4) | Mix | Quantity(100ml) | | --- | --- | | KOAc | 10 mM | | CaCl2.2H2O | 80 mM | | MnCl2.4H2O | 20 mM | | MgCl2.6H2O | 10 mM | | glycerol | 10% | 5]HEPES buffer(pH 7.4) | Mix | Quantity(100ml) | | --- | --- | | 10mM HEPES | 2.38g |

Transformation

###### | METHOD 1 | METHOD 2 | METHOD 3 | METHOD 4 | | --- | --- | --- | --- | | 1] Add 1ul of Ligated product in 50ul of competent cells | 1]Add 10ul of Ligated product in 50ul of competent cells | 1]Thaw JM109 on ice . Add 1ul DNA . Mix well . | 1]Dilute plamid 1:10. Add 10ul in 200ul. | | 2] Keep on ice for 30 min | 2] Keep on ice for 30 min | 2]Keep on ice for 30 min | 2]Keep on ice for 30 min | | 3]Heat shock for 45 sec at 42˚ C | 3]Heat shock for 45 sec at 42˚ C | 3]Heat shock for 30 sec at 42˚ C | 3]Heat shock for 2 minute at 42˚ C | | 4]Ice for 5 min | 4]Ice for 5 min | 4]Ice for 2 minutes | 4]Ice for 1 minute | | 5]Add 950 ul of SOC media. Incubate for 1 hour. | 5]Add 900 ul of LB media. Incubate for 1 hour. | 5]Add 400 ul of SOC . Incubate 1 hour. | 5]Add 400 ul of SOB . Incubate for 1 hour. | | 6]Spread plate and incubate. | 6]Spread plate and incubate. | 6]Spread plate and incubate. | 6]Spread plate and incubate. |

## **PROTOCOLS**

Gradient PCR

###### | Component | Amound for 25μl Reaction | | --- | --- | | 5X Flexi buffer | 5 μl | | dNTPs | 0.5 μl | | Forward Primer | 1 μl | | Reverse Primer | 1 μl | | Template DNA | 1 μL | | Taq DNA Polymerase | 0.2 μl | | Nuclease free water | To 50 μL | | MgCl2 | 0.2 μl | | Step | Temperature | Time | Cycles | | --- | --- | --- | --- | | Initial Denaturation | 98°C | 5 min | 1 | | Denaturation Annealing Extension | 95°C 64°C to 72°C 72°C | 20 sec 30 sec 30 sec | 30 | | Final Extension | 72°C | 10 min | 1 |

Gel Electrophoresis

###### 1. 1% Agarose gel (1gm in 100ml 1XTAE buffer) was melted. 2. EtBr was added and allowed to solidify. 3. 2ul loading dye with 10ul sample was loaded on gel. 4.Gel electrophoresis at carried out at 70V. 5. After loading dye reaches 70% of the length of the gel observe using gel doc.

Purification(Promega PCR cleanup kit)

###### 1. Add the remaining PCR product to a column mounted on a collection tube. 2. Spin at 10,000 rpm for 1 min. 3. Discard the contents in the collection tube. 4. Add 700µl of membrane wash solution to the column. 5. Spin at 16,000rpm for 1 min. 6. Discard the contents in the collection tube. 7. Add 500µl of membrane wash solution to the column. 8. Spin at 16,000rpm for 1 min. 9. Discard the contents in the collection tube. 10. Spin at 16,000rpm for 1 min to remove excess ethanol. 12. Add 20µl of nuclease free water. 13. Spin at 16,000rpm for 1 min. 14. Add 20µl of nuclease free water. 15. Spin 16,000rpm for 1 min. 16. Store 40µl of purified product in -20˚C.

Ligation

###### Concentration of DNA =OD(@260nm)\*50\*50 Ligation=(ratio\*kb of insert\*100)/(kb of vector) | Component | Amount for a 10 μl Reaction | | --- | --- | | T4 DNA Ligase Buffer with ATP (10x) | 1 μl | | Vector Backbone | 25-50ng | | Insert DNA | 25-100ng (3:1 molar ratio of insert:vector) | | T4 DNA Ligase | 0.5 μl | | ddH2O | To 10 μl |

Competent cells

###### 1. Inoculate JM109 in LB medium. Let it grow overnight. 2. Inoculate 1ml of overnight grown culture into fresh LB medium of 25ml. 3. Incubate for 3 hours. Check OD should be around 0.3. 4. Transfer this culture into centrifuge tubes of 2ml . 5. Spin at 15,000 rpm for 5 min at 4˚C. 6. Gently re-suspend in 500 μl of CaCl2 (10mM) and incubate in ice for 30 min. 7. Spin at 15,000rpm for 5 min at 4˚C. 9. Discard the supernatant and resuspend in 50 μl of CaCl2. 11. Incubate in ice for 2 hrs.

Transformation

###### 1. Add 1µl of ligated product in 50µl of competent cells. 2. Incubate on ice for 45 min. 3. Heat shock for 45sec on 42˚C. 4. Incubate in ice for 5 min. 5. Add 1 mL LB and incubate at 37˚C for 1.5 hour at 120-200rpm. 6. Centrifuge tube at 5,000rpm for 7 mins. 7. Resuspend the pellet in 100 μl supernatant at spread plate at chloramphenicol plates.

Colony PCR

###### | PCR mix | Amound for 25μl Reaction | | --- | --- | | 5X Flexi buffer | 10 μl | | dNTPs | 0.5 μl | | Forward Primer | 1 μl | | Reverse Primer | 1 μl | | plasmid | colony | | Taq DNA Polymerase | 0.4 μl | | Nuclease free water | To 25 μL | | MgCl2 | 2 μl | | Step | Temperature | Time | Cycles | | --- | --- | --- | --- | | Initial Denaturation | 95°C | 5 min | 1 | | Denaturation Annealing Extension | 95°C 54°C 72°C | 1 min 1.5 min 1min | 30 | | Final Extension | 72°C | 10 min | 1 |

Alkaline lysis

###### 1. Grow the transformants in 1 ml LB with appropriate antibiotic. 2. Take the culture and spin at 5,000rpm for 3 mins at 4˚C. 3. Re-suspend cell pellet in 50µl chilled solution I. 4. Incubate in ice for 5 min. 5. Add 100µl of solution II. Mix gently. 6. Incubate in ice for 10 min. 7. Add 75µl of solution III. Mix gently. 8. Incubate in ice for 15 min. 10. Centrifuge at 12,000rpm for 15 min at 4˚C. Solution I (pH 8): 1. 50mM glucose 2. 10mM EDTA 3. 25mM Tris-HCl Solution II : 1. 0.2% NaOH 2. 1% SDS Solution III: 1. 3M Potassium / sodium acetate

Plasmid Cleanup

###### 1. After alkaline lysis for plasmid isolation centrifuge at 12,000rp,for 15 min at 4˚C. 2. Treat the supernatant with 20µl of Rnase A. 3. Incubate at 37˚C for 30 min at 200 rpm. 4. Equal volume of phenol : chloroform : isoamyl alchohol was added. 5. Keep in ice for 10 min. 6. Spin at 10,000rpm for 10 min at 4˚C. 7. Collect the aqueous phase. 8. Add 150mM NaCl. Mix well. 10. Add 2ml of absolute chilled ethanol. Mix gently. 11. Incubate for 1 hour in -20˚C. 12. Spin at 10,000rpm for 15 min at 4˚C. 13. Wash with 70% ethanol. 14. Spin at 10,000rpm for 10 min at 4˚C. 15. Discard the supernatant. 16. Air dry. 17. Re-suspend in 20µl of autoclaved water.

SDS Page

###### Reagents : ~30% Acrylamide (pH 7): 1. Add 290g acrylamide. 2. 10g of N,N'-methylbisacrylamide in 600ml of distilled water. 3.Make up the volume to 1L with distilled water after adujsting the ph. (tip:Heating may be necessary to dissolve the acrylamide.) ~Bisacrylamide: 1.Add 29.22 g of acrylamide. 2.0.78 g of bisacrylamide. 3.Add 100 ml of distilled water. ~1.5M Tris-HCL(ph8.8): 1.Add 90.75g of Tris-free base. 2.Add 300ml water. 3.Add 8ml of concentrated HCL. 4.Adjust pH to 8.8 . 5.Make the volume upto 500ml. ~1M Tris-HCL (pH 6.8): 1.Add 60.54g of Tris-free base. 2.Add 300ml of water. 3.Add 36ml of concentrated HCL. 4.Adjust the pH to 6.8. 5.Make up the volume to 500ml. 1. Resolving gel : | Components | Amount | | --- | --- | | ddH2O | 1 mL | | 1.5 M Tris/HCl (pH 8.8) | 3.0 mL | | 30% A/C stock | 3.2 mL | | 10% w/v SDS | 120 µl | | 10% w/v APS | 120 µl | | TEMED | T5 µl | 2. Stacking gel: | Components | Amount | | --- | --- | | ddH2O | 3.6 mL | | 1.5 M Tris/HCl (pH 6.8) | 1.56 mL | | 30% A/C stock | 0.6 ml | | 10% w/v SDS | 20 µl | | 10% w/v APS | 140 µl | | TEMED | 5 µl | 3. Butanol : 50 mL ddH2O + 30mL butanol 4. 2X running buffer : For 8mL | Components | Amount | | --- | --- | | ddH2O | 2.8mL | | 0.5M Tris/HCl (pH 6.8) | 1 mL | | glycerol | 0.8 ml | | 10% w/v SDS | 3.2mL | | 0.2% bromophenol blue | 0.2mL | 5. 5X running buffer : For 8mL | Components | Amount | | --- | --- | | Tris-HCl pH 8.3 | 125mM | | Glycine | 1.23M | | SDS | 0.5% | 6. Staining and destaining solution: Coommassie G250= 0.5% Ethanol = 30% Acetic acid= 10% Procedure : 1.Assembling the glass plate Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer) down first, then place the shorter glass plate on top of it. Embed them into the casting frame and clamp them properly make sure that the bottom ends of the glass plates are properly aligned. If so place it on the casting stand. 2. Casting the gel Prepare 12% Resolving gel and 5% stacking gel using the composition given above. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. Pour the solution till the mark. Add a layer of water saturated butanol or distilled water on top of the gel so as to level the poured gel). Allow the gel to polymerize for 20-30 minutes. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Drain the water saturated butanol with strips of filter paper. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently.(Make sure you keep the comb ready by the side). Immediately place a comb in the stacking gel sandwich. Allow it to polymerize for at least 20 minutes. 3.Preparation of samples Samples were centrifuged at 6000 rpm for 10 min. Samples were then mixed with an equal volume of 2 x non reducing samples buffer. Boil for 10 mins, centrifuge at 5000 rpm for 5 min and 15 - 20 μl loaded per well. 10ul of denatured molecular weight markers were also electrophoresed on each gel. 4.Running the gel To assemble, take out the gels from the casting frame and clamp them in the gel apparatus (Make sure that the short plate always faces inside). When the plates are secured, place them in tank. Fill the inner chamber of the tank with buffer. Remove the comb carefully. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe a few times with distille water after loading. Attach the power supply by putting the lid. Set the voltage up to 150 V and run for 1 hour till the dye front reaches the end of the gel. 5. Gel washing Take out the gel and wash (incubate 1 hr at room temp on a rotating shaker to remove the SDS and renature your proteinases) with 2.5% TritonX100 in 50 mM tris pH 7.4, 5 mM CaCl2, 1 μM ZnCl2. Rinse briefly with deionized water. Incubate overnight at 37 °C with 50 mM Tris pH 7.4, 5 mM CaCl, 1 μM ZnCl2. 6.Staining and Destaining Stain with 0.5% Coomassie G250 for 30 minutes (entire gel should be dark blue) De-stain in 30% Ethanol/10% acetic acid until you see clear bands (a couple of minutes). Change to 2% acetic acid to stop staining. Rehydrate in 2% acetic acid overnight

Restriction Digestion

###### 1. RE mix for plasmid digestion : | Components | Amount (50 µl) | | --- | --- | | Nuclease free water | 41 µl | | Buffer (10X) | 5 µl | | BSA (100X) | 1 µl | | Plasmid | 2 µl | | RE | 1 µl | 2. RE mix for DNA insert digestion : | Components | Amount (50 µl) | | --- | --- | | Nuclease free water | 40 µl | | Buffer (10X) | 5 µl | | BSA (100X) | 1 µl | | DNA insert | 3 µl | | RE | 1 µl | 2. Incubate at 37˚C in hot water bath for 1 hour. **RE cleanup (Using Promega cleanup kit) :** 1. Binding solution (50 µl) + RE-DNA/plasmid mix (50 µl) 2. Put the mix in the column. Incubate for 1 min. 3. Centrifuge at 16,000 rpm for 1min. 4. Decant. Add 700µl of wash buffer. 5. Centrifuge at 16,000 rpm for 1min. 6. Decant. Add 500µl of wash buffer. 7. Centrifuge at 16,000 rpm for 1min. 8. Add the column in a new tube. Add 15 µl of nuclease free water in the column and incubate for 5 mins. 9. Centrifuge at 16,000 rpm for 1min. The supernatant has cleaned DNA.

Fluorescence microscopy

###### 1. Incubate the primary culture of potassium promoter with GFP and GFP with no promoter for 18 h at 37˚C in 2ml of Luria Broth. 2. Add different concentration of potassium in Luria Broth. 3. To this add 25ul of primary culture . 4. Incubate overnight for growth. 5. Make a glass slide with this culture. 6. Cover it with a cover slip. 7. Observe under fluorescent microscope.

## **ASSAYS**

Blood collection and analysing serum

###### Blood was collected in heparin tubes to prevent clotting , to collect equal amount of serum from 2 ml aliquots. Blood was centrifuged to collect serum to be used for analyzing the electrolyte present in it. This analyzing was done using electrolyte analyzer in Vishawaraj Hospital ,Loni. This is an important aspect of our project as we will be dealing with blood in the sanitary napkins. This provides us with the concentration of potassium which is required to induce our promoter in a particular range. Blood was also used to check the interaction with Laccase enzyme whether it or it does not effect the functionality.

Fluorescent measurement

###### This was also performed in presence of serum to check if there were any hinderance with the components of blood. 1.Inoculate primary culture in LB. 2.Grow it over night. 3.Take OD of the sample. 4.Calculate the culture to be taken to dilute it to 0.1 OD. 5.Wash three times with 0.8% NaCl. 6.Resuspend pellet in 2ml K0 media. 7.Take 25µl of suspended cells and inoculate in 2ml media of different concentration of KCl. 8.Incubate overnight and one more set for 2-3 hours. Reagent : K0 minimal media | Reagents | Working concentration | | --- | --- | | Na2HPO4 | 46 mM | | NaH2PO4 | 23 mM | | (NH4)2SO4 | 8 mM | | MgSO4 | 0.4 mM | Citric acid | 0.66 mM || Tri-sodium citrate dehydrate | 0.33 mM | | Thiamine hydrochloride | 1 mg/liter | | Glucose | 2 g/liter. | | Casein acid hydrolysate | 0.2% |

Laccase assay

###### Laccase enzyme are blue copper oxidases having versatile substrate like phenolics, aromatic amines, and other electron-rich substrates by reducing O2 to H2O. Laccase activity was determined by the oxidation of ABTS method. ABTS is oxidized by laccase to cation radical. The concentration of this radical is responsible for the intense blue-green color that shows enzyme activity which is to be read at 420 nm in a spectrophotometer. This assay was performed in presence of blood as well to check if Laccase is induced in presence of blood due to iron , as laccae being a metalo-enzyme ; co factor as copper . 1]Make a mixture of 200ul of 0.5 mM ABTS,1.5ml 0.1 M sodium acetate (pH 4.5), add 100 μL of culture and 200ul of supernatant 2]Incubate for 5 min. 3]Absorbance was read at 420 nm in a spectrophotometer against a suitable blank.

Crystal violet assay

###### 1]Biofilms were grown by subculturing 18 h cultures to a starting OD600 of 0.2 and immediately diluting them 1:100 in BHI supplemented with 0.4% glucose. 2]A 5 ml aliquot of each strain was added to each of the 3 wells. 3]Biofilms were allowed to shake at 100 rpm at 37°C for 18 h. To harvest the biofilms, the media was aspirated off and the biomass was washed twice with 5 ml of sterile water. 4] The biomass was allowed to air dry and stained for quantification. 5]To assess biomass, biofilms were stained with 500 μl of 0.1% crystal violet for 10 min at room temperature. 6]Excess dye was removed and biofilms were washed with 5 ml of sterile water. 7]Add 200ul acetic acid and read absorbance at 580nm.

Cover slip assay

###### 1]Biofilms were grown by subculturing 18 h cultures to a starting OD600 of 0.2 . 2]These subcultures were then incubated with a cover slip in with luria broth and crystal violet. 3]These slips were washed . 4] Visualization was observed.

Made with ❤ in India

![140x140](https://static.igem.org/mediawiki/2019/7/74/T--MITADTBIO_Pune--MITlogo.png)

##### LINKS

[MIT SBSR](https://www.mitbio.edu.in)

[MIT ADT University](http://www.mituniversity.edu.in/)

##### ADDRESS

MIT School of Bioengineering, Sciences and Research, Pune, Maharashtra 412201

##### CONTACT

Ashima Khanna

Team Lead

igem.mitadtbio.pune@gmail.com

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

# Team:MITADTBIO Pune/Results

* [**HOME**](https://2019.igem.org/Team%3AMITADTBIO_Pune)* **TEAM▾**
    + [**| TEAM MEMBERS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Team)
    + [**| COLLABORATIONS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Collaborations)
    + [**| ATTRIBUTIONS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Attributions)
  * **PROJECT▾**
    + [**| DESCRIPTION**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Description)
    + [**| DESIGN**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Design)
    + [**| EXPERIMENTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Experiments)
    + [**| RESULTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Results)
    + [**| MATHEMATICAL MODELLING**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Model)
    + [**|FUTURE PROSPECTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/3D%20MODEL)
  * [**PARTS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Basic_Part)
  * [**SAFETY**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Safety)
  * **COMMUNITY▾**
    + [**| HUMAN PRACTICES**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Human_Practices)
    + [**| PUBLIC ENGAGEMENT**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Public_Engagement)
    + [**| SURVEY**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Survey)
  * **AWARDS▾**
    + [**| MEDALS**](https://2019.igem.org/Team%3AMITADTBIO_Pune/Medal)

![](https://static.igem.org/mediawiki/2019/6/67/T--MITADTBIO_Pune--resultsheader.png)

**[KdpF promoter](#target1)**

**[Laccase](#target2)**

[**Biofilm**](#target3)

**KdpF PROMOTER**

The aim of this project is to create a genetically engineered organism that has the capability of releasing extracellular degradation enzymes to deteriorate polyethylene (PE) based sanitary pads. PEred’s success hinges on it detecting K+ levels in its surroundings and using this as a trigger to express a plastic degrading enzyme. To facilitate this, we decided to construct reporter strain to express green fluorescence protein (GFP) using a potassium inducible promoter that is active in presence of menstrual blood.
The potassium promoter sequence used was based on the design BBa\_ K1682002 created by iGEM15\_HKUST-Rice. We got potassium promoter with modified *EcoRI* sites synthesized from DeNovo Tech India, which was used as template to amplify the promoter fragment using following primers:

Kdp\_F: ATCAGAATTCTGCCATTTTTATACTTTTTT
Kdp\_R: CTGAGAATTCCAGAAATCTACCCTTCCGGT

![thumbnail1](https://static.igem.org/mediawiki/2019/0/04/T--MITADTBIO_Pune--KdpFPCR.png)

Figure 1. Construction of KdpF promoter in GFP plasmid. Agarose gel electrophoresis of DNA fragments obtained from PCR amplification using Kdp-F and Kdp-R primers 1. Potassium promoter 2. Colony PCR of BBa\_K3306002 recombinant plasmid 3. Colony PCR of BBa\_E0040. M: 100 bp DNA ladder.

PCR amplification of potassium promoter using forward and reverse primers gave an expected 78 base pair DNA band (Figure 1, Lane 1)
As the aim of using KdpF promoter was to control the expression of downstream genes using potassium, it was necessary to quantify the induction of KdpF potassium promoter in *E. coli*. To construct reporter strains, our PCR amplified promoters was cloned upstream of GFP derived from *Aequeora Victoria* [BBa\_E0040]to construct composite part[BBa\_K3306002](http://parts.igem.org/Part%3ABBa_K3306002). The resultant plasmid was then transformed in *E. coli* JM109 competent cells. To check the correct insertion of the fragment, few colonies were sub-cultured and used to extracted plasmids. Colony PCR of the isolated recombinant plasmid was performed using KdpF promoter specific forward and reverse primers gave expected band of 78 bp confirms that potassium promoter is successfully integrated into BBa\_E0040 (Figure 1, Lane 2, 3)

#### Characterizing our promoter repertoire

To establish whether reporter strains could successfully detect the presence of potassium ions, we did an initial fluorescence microscopy test with the reporter strain incubated with 0.5mM potassium.
Figure 2 shows some of the images from this test, where it can be seen that incubation of our reporter strain with potassium leads to increase GFP expression (Figure 2B) compared to basal GFP expression levels in cells grown in absence of potassium. As seen in Figure 2A, transformants grown in the absence of potassium shows very/low basal luminescence level most likely from promoter leaking. *E. coli* cells alone were used as a negative control (Figure 2C). This result demonstrates that the KdpF promoter is functional and that we successfully created reporter *E.coli* strain containing GFP under the control of KdpF promoter. This recombinant is able to express GFP using potassium ions as inducible molecules and thus validate our part.

![thumbnail1](https://static.igem.org/mediawiki/2019/d/dd/T--MITADTBIO_Pune--control.png)

![thumbnail1](https://static.igem.org/mediawiki/2019/8/80/T--MITADTBIO_Pune--induced.png)

![thumbnail1](https://static.igem.org/mediawiki/2019/f/f6/T--MITADTBIO_Pune--uninduced.png)

Figure 2. Fluorescence Microscopy. A. Control - not induced; B. Test induced - induction with potassium; C. Negative control

To determine the minimum and maximum concentration of potassium required to induce GFP expression, the reporter strain was further analyzed with a dose-dependence study. Transformants with our composite part BBa\_K3306002 were grown on LB media. The OD600 of the cells was measured and equalized to 0.1 OD to ensure comparability. The cells were diluted and relative concentration of GFP was recorded in Tecan Infinite m200 fluorescence spectrophotometer at 600nm.
The graph in figure 4 clearly indicates that at different concentrations of potassium, there is also an increase in the expression of GFP, but it is not linear in nature.

![thumbnail1](https://static.igem.org/mediawiki/2019/d/de/T--MITADTBIO_Pune--potassium.png)

Figure 3. Changes in fluorescence with varying potassium ion concentration.

Since the goal of our system is to enable engineered bacteria to sense and trigger the expression of the downstream gene in presence of potassium ions present in **blood**, after standardizing our part BBa\_K3306002 in KCl, we wanted to test its activity in blood. Blood serum was collected as described in protocols. The potassium ion concentration in serum was analyzed using Microlyte electrolyte analyzer.

###### | Electrolyte | Concentration | | --- | --- | | K+ | 4.3 mM | | Na+ | 14.3 mM | | Cl- | 110 mM |

Through the implementation point of view, we decided to evaluate the performance of potassium promoter in presence of blood serum as a source of potassium ions to simulate the conditions in our proposed bioreactor that PEred would be exposed to in the future. This is achieved by growing our transformants in different K+ concentrations which are obtained from diluting serum in LB. Figure 5 suggests that the KdpF promoter starts expressing GFP at different concentrations wherein elevated levels of expression was found at the concentration of 1.7mM. But this was not significantly high compared to remaining potassium concentrations. This clearly indicates that the induction of GFP expression by KdpF promoter remained insensitive to the variation in potassium concentration in serum. We can hypothesize that due to high osmolarity of serum, the expression of Kdp pathway becomes independent of potassium in the environment. This hypothesis would have to be validated with further experiments.

![thumbnail1](https://static.igem.org/mediawiki/2019/2/21/T--MITADTBIO_Pune--serum.png)

Figure 4. Emission of reporter strain with BBa\_K3306002 in presence of serum.

---

**LACCASE**

The enzyme *laccase* is an important part of our genetically engineered organism, as it catalyzes polyethylene deterioration.
The iGEM DNA kit include few *laccase* enzymes such as BBa\_K863000, BBa\_K863001, BBa\_K863005, BBa\_K863006, BBa\_K863010, BBa\_K863011, BBa\_K863020, BBa\_K863021, BBa\_K729002, BBa\_K808025 and BBa\_K729002. However except BBa\_K808025 (Cutinase) and BBa\_K729002 (laccase), all the other *laccase* have applications concerning bioremediation, detoxification of industrial effluents and wastewater purification.
Since we will be using *laccase* to degrade the plastic component of our used menstrual pads, only two genes qualify for this requirement. *Cutinase* is well recognized for stimulating Polyethylene terephthalate (PET) hydrolysis and degradation[1].
On the other hand *laccase*, has been demonstrated to effectively degrade polyethylene[2] and nylon-66[3] by oxidation of the hydrocarbon backbone of polyethylene. Between the two, *laccase* was selected to be part of our composite part, as sheet of conventional sanitary napkins is made from polyethylene (PE) and polypropylene (PP).

![thumbnail1](https://static.igem.org/mediawiki/2019/1/16/T--MITADTBIO_Pune--PET.png)

![thumbnail1](https://static.igem.org/mediawiki/2019/b/bb/T--MITADTBIO_Pune--laccase.png)

Figure 1A. BBa\_K808025 and 1B. BBa\_K729002

Cloning of *laccase* in iGEM backbone plasmid will lead to normal low level of *laccase* expression which will show *in vitro* activity, but the activity and expressive rate is non-obvious. For the above reasons, we decided to clone laccase gene is pET28a+ expression vector for sustainable production of *laccase*.
To insert *laccase* gene into cloning expression vector (pET 28a+), we first designed prefix and suffix primers which contain *EcoRI* and *PstI* restriction enzyme sites respectively. The PCR amplification of *laccase* gene from BBa\_K729002 using *EcoRI*-prefix and *PstI*-suffix primers give an expected 1.5 kb DNA band as shown in (Fig. 2).
Primers used to amplify laccase :

*EcoRI*\_Prefix\_F: ATGAGAATTCGTTTCTTCGAATTCGCGGCCGCTTCTAG
*PstI*\_Suffix\_R: GACTCTGCAGGTTTCTTCCTGCAGCGGCCGCTACTAGTA

![thumbnail1](https://static.igem.org/mediawiki/2019/f/fd/T--MITADTBIO_Pune--ORF.png)

Figure 2. PCR amplification of *laccase* ORF using prefix and suffix primers gave expected size DNA band. M: 500 bp DNA ladder; 1: PCR amplified laccase gene (size 1.5 kb).

The purified PCR amplified products were restriction double digested with *EcoRI-PstI*, and ligated at same sites in pET28a+ (5.3 kb) expression vector and transformed recombinant plasmid into *E. coli* (JM109). The transformation result was shown in Figure 3. To check whether the construction was successful, we picked few colonies to extract their plasmids. The inclusion of insert into the recombinant plasmid was verified both by restriction digestion (Fig 3), and DNA sequencing. Our sequencing results with the prefix and suffix primers gave mixed results. The data obtained from sequencing results was aligned to the original biobrick. Since the sequences showed 97% match, this confirmed the insert was successfully cloned and was in correct orientation.

![thumbnail1](https://static.igem.org/mediawiki/2019/4/46/T--MITADTBIO_Pune--pet1.png)

Figure 3. Restriction digestion of *laccase* carrying recombinant plasmid using *EcoRI*. M: 500 bp DNA ladder; 1: Plasmid showing expected size band of 1.55 kb.

We tested the expression of *laccase* enzyme by transforming the pET28a+ recombinant carrying pET-laccase gene into *E. coli* expression strain BL21. To overexpress *laccase* enzyme, *E. coli* expression strain BL21 containing *laccase* gene under IPTG-inducible T7 promoter was induced by 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3hrs and 18 hrs at 37°C, corresponding proteins were separated by 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue G250 (CBB). Prior to induction with IPTG, expression of *laccase* is not detectable by SDS-PAGE (Figure 4 Lane 1). After IPTG induction recombinant, protein bands of about 58 kDa was observed which is in agreement with the theoretical molecular weight of laccase which is 58.87 kDa (Figure 4 Lane 2).

![thumbnail1](https://static.igem.org/mediawiki/2019/0/02/T--MITADTBIO_Pune--SDS.png)

Figure 4. Induction and overexpression of recombinant protein: Samples were subjected to 12% SDS-PAGE and stained with Commassie Brilliant Blue G250. A- Lane 1, total cellular proteins from *E. coli* BL21(DE3) transformed with pET-laccase uninduced; Lane 2, total cellular proteins from *E. coli* BL21(DE3) transformed with pET-laccase induced by IPTG for 3 hrs; Lane 2, total cellular proteins from *E. coli* BL21(DE3) transformed with pET-laccase induced by IPTG for 18 hrs.

#### *IN VITRO* ACTIVITY ASSAY OF LACCASE

The above gel confirms that the construct is well assembled and successfully synthesize the desired *laccase* protein. Then, we tested whether expressed laccase is active through enzyme activity experiments. To detect *laccase* enzyme activity, colorimetric assay was carried out using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS method.
The nonphenolic dye ABTS is oxidized by *laccase* into water soluble chromogen ABTS+ which is more stable and preferred state of the cation radical. ABTS+ can be monitored spectrophotometrically at 420 nm. The concentration of the cation radical responsible for the intense blue-green color can be correlated to enzyme activity and is read at 420nm.
The supernatant of overnight grown bacteria culture acted as a source of *laccase* enzyme. The enzyme activity experiment was carried out by using 100 μL of enzyme and 100 μL of ABTS solution (0. 5 mM) in sodium acetate buffer (pH 5). The enzymatic units (U) defined as the amount of enzyme transforming 1 µmol of substrate per minute is calculated by the following formula:
Equation : U/L = (∆E×Vt)/(ε×d×Vs)
with ∆E being the change in extinction of light [min-1] at 420 nm
 ε being the molar absorption coefficient of ABTS [M-1 cm-1]
 d being the layer thickness [cm] in your cell that the light has to pass
 Vt is the total volume measured and Vs is the volume of the enzyme stock solution added to the ABTS stock solution.
It is demonstrated in Figure 5 by *in vitro* ABTS assay that recombinant *laccase* protein can oxidize ABTS. It is essential for our project that recombinant microorganisms should be capable of polyethylene deterioration in the presence of blood and/or dried blood. To check the effect of blood on *laccase* enzyme activity, we performed the ABTS assay in the presence of different concentrations of blood. It was clearly observed that the *laccase* activity is not significantly affected by blood (Figure 5).

![thumbnail1](https://static.igem.org/mediawiki/2019/d/d0/T--MITADTBIO_Pune--plot.png)

Figure 5: *Laccase* activity in presence and absence of blood. The fold differences were calculated compared with control groups and shown in the figure. \*P<0.007; \*\*P<0.01; [one-way analysis of variance].

We were not only able to successfully clone the *laccase* gene into an expression vector (Figure 3), but we were also able it induces its expression (Figure 4). Finally, we could also show the *laccase* activity *in vitro* as well as the influence of blood on *laccase* enzyme activity (Figure 5).

---

**BIOFILM**

Literature survey suggests that biofilm formation on the surfaces of plastic films was important for its degradation[4,5] as well as we wanted to engineer PEred such that it spread across the waste thus rapidly increasing biomass which would eventually lead to rapid degradation of plastic.
The biofilm formation regulator OmpR234 gene from BBa\_K342003 was inserted into cloning vector (pGEMT) using strategy as described earlier for *laccase* gene. Briefly OmpR234 gene were PCR amplified from using *PstI* prefix and suffix primers. After performing clean up of PCR amplified products, genes were blunt end ligated to vector and transformed recombinant plasmid into *E. coli* (JM109). The transformants were ascertained by PCR amplification using gene specific primers. PCR using *PstI* primers prefix suffix primers gives a 651 bp DNA fragment corresponding to intact OmpR234 gene when recombinant plasmid DNA was used as the template.
Primers used to amplify biofilm:

*PstI*\_Prefix\_F: ATGACTCCAGGTTTCTTCGAATTCGCGGCCGCTTCTAG
*PstI*\_Suffix\_R: GACTCTGCAGGTTTCTTCCTGCAGCGGCCGCTACTAGTA

![thumbnail1](https://static.igem.org/mediawiki/2019/7/75/T--MITADTBIO_Pune--biofilmgel.png)

Figure 1. PCR amplification of biofilm inducing gene. M: 500bp DNA ladder, 1: 651 bp OmpR234 gene PCR product.

To ascertain the adherence property of the recombinant cells, we used a coverslip based technique[6]. As seen in Fig. 2, after 18 h of growth, recombinant bacteria formed thick and confluent biofilm structures on the coverslip detected after staining with 0.1% crystal violet. The test revealed dark, confluent staining across the surface indicating robust biofilm formation. One representative picture is presented here.

![thumbnail1](https://static.igem.org/mediawiki/2019/3/33/T--MITADTBIO_Pune--cover.png)

Figure 2. Visualization of coverslip biofilm in A. Control and B. *E. coli* with OmpR234 gene. The coverslips were post stained with crystal violet and images were taken.

#### CRYSTAL VIOLET ASSAY

Crystal violet assay was used to measure the biofilm formation capabilities of the transformants and its quantification using microplate method. As illustrated in Figure 3, the noticeable difference between recombinant cell carrying OmpR234 gene as compare to control show distinct extracellular biofilm production in the modified strain.

![thumbnail1](https://static.igem.org/mediawiki/2019/f/ff/T--MITADTBIO_Pune--biolfimtable.png)

Figure 3. Crystal violet assay of transformants. Blank LB; Control: E. coli cells; Recombinant: E. coli cells carrying OmpR234 gene

The crystal violet staining visually illustrates that the expression of OmpR234 in our experiments leads to about 3 times more biofilm compared to the control. The recombinant also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps.

#### **References**

1. H.P. Austin, M.D. Allen, B.S. Donohoe, N.A. Rorrer, F.L. Kearns, R.L. Silveira, B.C. Pollard, G. Dominick, R. Duman, K. El Omari, V. Mykhaylyk, A. Wagner, W.E. Michener, A. Amore, M.S. Skaf, M.F. Crowley, A.W. Thorne, C.W. Johnson, H.L. Woodcock, J.E. McGeehan, G.T. Beckham, Characterization and engineering of a plastic-degrading aromatic polyesterase, Proc. Natl. Acad. Sci. (2018) 201718804. doi:10.1073/pnas.1718804115.
2. Bhardwaj, H., Gupta, R., and Tiwari, A. (2013) Communities of microbial enzymes associated with biodegradation of plastics. J Polym Environ 21: 575–579.
3. M. Fujisawa, H. Hirai, T. Nishida, Degradation of polyethylene and nylon-66 by the laccase-mediator system, J. Polym. Environ. 9 (2001) 103-108.
4. Morgan Vague1, Gayle Chan2, Cameron Roberts1, Natasja A. Swartz2 and Jay L. Mellies1, 2019.Pseudomonas isolates degrade and form biofilms on polyethylene terephthalate (PET) plastic. BioRxiv preprint. doi: https://doi.org/10.1101/647321.
5. Balasubramanian, V. et al. High-density polyethylene (HDPE)-degrading potential bacteria from marine ecosystem of Gulf of Mannar, India. Lett ApplMicrobiol 51, 205211, doi:10.1111/j.1472-765X.2010.02883.x (2010).
6. Walker JN, Horswill AR. 2012. A coverslip-based technique for evaluating Staphylococcus aureus biofilm formation on human plasma. Front. Cell. Infect. Microbiol. 2:39