iGEM 2019 — Team ITB_Indonesia

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# Team:ITB Indonesia/Notebook

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* Team

  [Team](https://2019.igem.org/Team%3AITB_Indonesia/Team)
  [Attributions](https://2019.igem.org/Team%3AITB_Indonesia/Attributions)
  [Collaborations](https://2019.igem.org/Team%3AITB_Indonesia/Collaborations)
  [Gallery](https://2019.igem.org/Team%3AITB_Indonesia/Gallery)
* Project

  [Description](https://2019.igem.org/Team%3AITB_Indonesia/Description)
  [Design](https://2019.igem.org/Team%3AITB_Indonesia/Design)
  [Experiments](https://2019.igem.org/Team%3AITB_Indonesia/Experiments)
  [Safety](https://2019.igem.org/Team%3AITB_Indonesia/Safety)
  [Model](https://2019.igem.org/Team%3AITB_Indonesia/Model)
  [Hardware](https://2019.igem.org/Team%3AITB_Indonesia/Hardware)
* Parts

  [Parts](https://2019.igem.org/Team%3AITB_Indonesia/Parts)
  [Part Collection](https://2019.igem.org/Team%3AITB_Indonesia/Part_Collection)
* Results

  [Results](https://2019.igem.org/Team%3AITB_Indonesia/Results)
  [Demonstrate](https://2019.igem.org/Team%3AITB_Indonesia/Demonstrate)
* Human

  [Human Practices](https://2019.igem.org/Team%3AITB_Indonesia/Human_Practices)
  [Integrated Human](https://2019.igem.org/Team%3AITB_Indonesia/Integrated_Human)
  [Public Engagement](https://2019.igem.org/Team%3AITB_Indonesia/Public_Engagement)
* Awards

  [Medals](https://2019.igem.org/Team%3AITB_Indonesia/Medals)
  [Awards](https://2019.igem.org/Team%3AITB_Indonesia/Awards)
* [Main Sponsor](https://2019.igem.org/Team%3AITB_Indonesia/MainSponsor)

[![](https://static.igem.org/mediawiki/2019/f/f5/T--ITB_Indonesia--logo_black.png)](https://2019.igem.org/Team%3AITB_Indonesia)
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# 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:ITB Indonesia/Experiments

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* Team

  [Team](https://2019.igem.org/Team%3AITB_Indonesia/Team)
  [Attributions](https://2019.igem.org/Team%3AITB_Indonesia/Attributions)
  [Collaborations](https://2019.igem.org/Team%3AITB_Indonesia/Collaborations)
  [Gallery](https://2019.igem.org/Team%3AITB_Indonesia/Gallery)
* Project

  [Description](https://2019.igem.org/Team%3AITB_Indonesia/Description)
  [Design](https://2019.igem.org/Team%3AITB_Indonesia/Design)
  [Experiments](https://2019.igem.org/Team%3AITB_Indonesia/Experiments)
  [Safety](https://2019.igem.org/Team%3AITB_Indonesia/Safety)
  [Model](https://2019.igem.org/Team%3AITB_Indonesia/Model)
  [Hardware](https://2019.igem.org/Team%3AITB_Indonesia/Hardware)
* Parts

  [Parts](https://2019.igem.org/Team%3AITB_Indonesia/Parts)
  [Part Collection](https://2019.igem.org/Team%3AITB_Indonesia/Part_Collection)
* Results

  [Results](https://2019.igem.org/Team%3AITB_Indonesia/Results)
  [Demonstrate](https://2019.igem.org/Team%3AITB_Indonesia/Demonstrate)
* Human

  [Human Practices](https://2019.igem.org/Team%3AITB_Indonesia/Human_Practices)
  [Integrated Human](https://2019.igem.org/Team%3AITB_Indonesia/Integrated_Human)
  [Public Engagement](https://2019.igem.org/Team%3AITB_Indonesia/Public_Engagement)
* Awards

  [Medals](https://2019.igem.org/Team%3AITB_Indonesia/Medals)
  [Awards](https://2019.igem.org/Team%3AITB_Indonesia/Awards)
* [Main Sponsor](https://2019.igem.org/Team%3AITB_Indonesia/MainSponsor)

[![](https://static.igem.org/mediawiki/2019/f/f5/T--ITB_Indonesia--logo_black.png)](https://2019.igem.org/Team%3AITB_Indonesia)
![](https://static.igem.org/mediawiki/2019/7/7f/T--ITB_Indonesia--kai_logo.png)
![](https://static.igem.org/mediawiki/2019/e/e0/T--ITB_Indonesia--itb_logo.png)

![](https://static.igem.org/mediawiki/2019/c/cc/T--ITB_Indonesia--header-experiments.png)

### Protocols

#### [Making chemically competent cells](#collapseOne)

##### Materials :

* *Escherichia coli* TOP10 Culture
* LB agar plate
* Super Optimum Broth (SOB)
* CCMB80 buffer (from: [http://parts.igem.org/Help:Protocols/Competent\_Cells)](http://parts.igem.org/Help%3AProtocols/Competent_Cells)
* SOB (from : http://parts.igem.org/Help:Protocols/SOB)
* Ice bucket
* 50 mL and 15 mL Falcon
* 100 mL Erlenmeyer Flask
* 1.5 microcentrifuge tube

##### Protocol :

1. Streak *E.coli* from glycerol stock to LB plate
2. Incubate the plate overnight (16-18 hrs)
3. Inoculate 5 mL SOB in 15 mL sterile Falcon with *E.coli* from the
   plate, then incubate
   it overnight in 37oC with continuous shaking at 200 rpm
4. Inoculate 47.5 mL SOB in 100 mL sterile Erlenmeyer with 2.5 mL overnight
   *E.coli*.
   Incubate it in 37oC with continuous shaking at 200 rpm until it
   reach
   OD600 0.3-0.4
   (usually 20-30 minutes for TOP10)
5. Aliquot @15 mL culture to 15 mL sterile Falcon, then centrifuge it at 3000g,
   4oC,
   for 10 minutes. Discard the supernatant
6. Resuspend the cell pellet with 8 mL ice cold CCMB80 buffer and incubate it
   on ice
   for 20 minutes
7. Centrifuge the resuspended culture at 3000 g, 4oC, for 10
   minutes.
   Discard the
   supernantant
8. Resuspend the pellet with 1 mL Ice cold CCMB80 buffer and incubate it for 20
   minutes
9. Aliquot the chemically competent cell @50 µL to 1.5 mL microcentrifuge tube
   and
   store it in -80oC until usage

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Transformation](#collapseTwo)

##### Materials :

* *Escherichia coli* TOP10 chemically competent cells
* LB agar plate with appropriate antibiotic
* SOC (SOB with 20 mM glucose,from : <http://parts.igem.org/SOC>)
* 1.5 mL microsentrifuge tube

##### Protocol :

1. Chill the chemically competent *E.coli*coli TOP10 culture (in 50 µL
   aliquot) in ice bucket
   for 10 minutes
2. Add 1-3 µL plasmid to the aliquot and incubate it on ice bucket for 30
   minutes
3. Incubate it on waterbath with 42oC temperature for 1 minute
4. Immediately move the tube to ice bucket and incubate for 5 minutes
5. Add 200 µL SOC to the tube and incubate it for 2 hours in 37oC,
   200 rpm
6. Innoculate the transformed culture @100 µL to LB plate with appropriate
   antibiotic,
   with spread plate method for easier single colony pick
7. Incubate the inoculated plate overnight in 37oC

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [PCR/Colony PCR](#collapseThree)

##### Materials :

* Dream Taq Green DNA Master Mix (2x) (Thermo Scientific)
* Nuclease Free Water
* Appropriate primer pair
* PCR tube
* Filtered tips
* Ice block

##### Protocol :

1. For Colony PCR, prepare sample by dilluting picked colony in 50 µL NFW
2. On ice, add PCR components (Dream Taq DNA polymerase, primer, NFW, and
   template)
   to make
   10 µL reaction

| Components | Volume (µL) |
| --- | --- |
| Dream Taq Green DNA Master Mix (2X) | 5 µL |
| F primer | 1 µL |
| R primer | 1 µL |
| NFW | 1 µL |
| Template | 2 µL |

3. Vortex mix and spin down all reactions
4. Put all samples in Thermal cycler and set the reactions as the table below

| Reaction | Temperature (oC) | Time | Number of Cycle(s) |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 3 min (colony: 4 min) | 1 |
| Denaturation | 95 | 30 seconds | 25-40 |
| Annealing | Appropriate temp | 30 seconds |
| Elongation | 72 | 1 min/lb |
| Final Extension | 72 | 5-15 min | 1 |
| Storage | 4 | - | - |

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* Filtered tips used for PCR preparation and performed in laminar air flow
  cabinet
  to
  prevent contamination in reagen
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [DNA ligase reaction](#collapseFour)

##### Materials :

* T4 DNA ligase (NEB)
* T4 DNA ligase buffer (NEB)
* 1.5 mL microtubes

##### Protocol :

###### Ligation reactions were done following NEB protocol (from : <https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202>)

1. To make 20 µL reactions, mix 2µL T4 DNA ligase with 1:3 molar ration of
   vector
   to
   insert, NFW to 20µL, and 1µL T4 DNA ligase. Mixing performed with the tube
   placed on
   ice.
2. Mix all reagents by pipetting up and down, continued with spin down
3. Incubate reactions in 37oC overnight
4. Heat inactivate at 65oC for 10 minutes and chill the reactions on
   ice
   for transformation
   into competent cells

##### Safety Considerations :

* Wearing personal protective safety equipment is necessary
* Keep working area clean and tidy
* Follow necessary handling and disposal considerations by the manufacturer

#### [Agarose gel electrophoresis](#collapseFive)

##### This protocol was performed to check the restriction reactions, PCR and plasmid isolation.

##### Materials :

* Agarose powder
* TAE buffer 1X (Tris, Acetate, EDTA)
* Loading dye 6X
* DNA ladder 1Kb
* EtBr (Ethidium Bromide)
* ddH2O

##### Protocol :

1. Dissolve 1g in 100 mL TAE 1X for 1% agarose and heat the mixture in
   microwave
   for 1-3
   minutes or until the mixture looked clear
2. Pour agar mixture in the gel cast and place a well comb. Wait until the gel
   solidify
3. Place gel in electrophoresis chamber filled with 1X TAE buffer
4. Insert mixture of DNA ladder and loading dye, and insert all samples (add
   loading dye if
   the sample doesn’t include one)
5. Run electrophoresis at 70V, for about 30-40 minutes
6. Visualize the DNA by staining the gel with EtBr solution, and destain it
   before
   observation
7. Place the gel on UV transilluminator

##### Safety Considerations :

* Wear personal protective safety equipment for performing this protocol
* EtBr is a toxic mutagen, use double protection e.g double gloves
* Used agarose gel disposed as cytotoxic waste

#### [*Escherichia coli* culture](#collapseSix)

##### Media :

###### We used Luria bertani media for all *E.coli* cultivation.

##### Materials :

* Tryptone (1%)
* Yeast Extract (0.5%)
* NaCl (1%)
* For Solid media, Agar A (2%)
* Distilled water

##### Media preparation :

###### To make LB media, mix all ingredients and dissolved them in distilled water. We usually pre heat the agar media solution in hotplate.

###### All LB media must be sterilized by autoclave.

##### For *E.coli* culture in solid media :

###### We usually inoculate *E.coli* in solid media using two methods namely streak and spread plate. Streak method used for rejuvenating culture, while spread plate used for spread the culture evenly in the agar plate so the single colonies are formed.

###### Spread plate: 100µL culture inoculated to the surface of solid LB media then spreaded using sterilized spreader

###### Streak: colonies are picked using sterile inoculating loop and gently scratched to agar plate

##### For *E.coli* culture in liquid media :

###### If the inoculum is liquid, we used micropipette equipped with sterilized tips to transfer the inoculum into fresh media. If the inoculum is grown in the solid media, we picked the colonies using inoculating loop and dissolve them in the fresh media. We used 200 rpm and 37oC as a default condition for growing *E.coli*.

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Vibrio parahaemolyticus standard curve](#collapseSeven)

###### To convert OD600 measurement into cell number, the standard curve is needed.

##### Materials :

* *V.parahaemolyticus* culture
* Sea water LB broth media (minus NaCl, dissolved in sea water)
* Sea water LB solid media
* Sea water

##### Protocol :

1. V.parahaemolyticus on solid media are inoculated to sea water LB broth media
   using
   inoculating loop. Culture are then incubated 24 hours at room temperature
   (RT)
   with 150
   rpm agitation
2. 24 hrs V.parahaemolyticus culture are inoculated to fresh sea water LB broth
   (10%) and
   incubated for 24 hrs again at RT, 150 rpm.
3. 10% culture are inoculated to fresh sea water LB broth and incubated for 12
   hrs
   at
   RT,150 rpm.
4. After 12 hrs, aliquot the culture to some falcon tubes and set the
   OD600 on
   each falcon different by diluting them in fresh sea water LB broth. Aim for
   OD600 0.2,
   0.4, 0.5, 0.6 and 0.7.
   Perform serial dilution to each cell culture in
   the
   falcon
   with 10X dilution on each transfer. Culture with dilution factor 10-4, 10-5,
   and
   10-6
   are inoculated to sea water LB solid media using spread plate method
5. Incubate the plate in 37oC for 24-48 hrs and count the colonies
   that
   appear on the plate
   in the range of 30-300, multiply with 10 and dilution factor.
6. Plot the OD600 to cell numbers

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Vibrio parahaemolyticus growth curve](#collapseEight)

###### To get the information of *V.parahaemolyticus* growth in the flask, we make its growth curve by measuring its OD600¬ every 2 hours for 14 hours.

##### Materials :

* *V.parahaemolyticus* culture
* Sea water LB broth media (minus NaCl, dissolved in sea water)
* Erlenmeyer Flask

##### Protocol :

1. Inoculate V.parahaemolyticus on solid media to sea water LB broth media
   using
   inoculating loop. Incubate the culture for 24 hours at room temperature (RT)
   with 150
   rpm agitation
2. 24 hrs V.parahaemolyticus culture are inoculated to fresh sea water LB broth
   (10%) and
   incubated for 24 hrs again at RT, 150 rpm.
3. Inoculate 10% culture to fresh sea water LB broth and incubate for 12 hrs at
   RT,150 rpm.
4. Inoculate 10% culture to fresh sea water LB broth and incubate at RT,150 rpm
   for
   14 hrs
   with OD600 measurement every 2 hrs using spectrophotometer
   UV-Vis.
5. Convert the OD600 data to cell numbers and plot the growth over
   time

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Plasmid Isolation : PrestoTM Mini Plasmid Kit](#collapseTen)

###### Plasmid DNA Isolation was carried out using the commercial kit from Geneaid Biotech Ltd. with the following instructions explained below.

##### Materials :

###### Equipment :

* Microcentrifuge
* Pipettes (1000 µL and 100 µL)

###### Consumables :

* PrestoTM Mini Plasmid Kit
* 1.5 mL microcentrifuge tubes
* Pipette tips (1000 µL and 100 µL)

##### Protocol :

1. Use 1.5-7.0 ml of cultured bacterial cells, pellet cells in a
   microcentrifuge
   for 1
   minute at 14,000-16,000 g. Discard the supernatant.
2. Add 200 µL of PD1 Buffer (make sure RNAse A was added). Resuspend the cell
   pellet
   completely by pipetting.
3. Add 200 µL of PD2 Buffer. Resuspend sample then mix gently by inverting the
   microcentrifuge tube 10 times. Let stand at room temperature for at least 2
   minutes to
   ensure the lysate is homogenous. Do not exceed 5 minutes
4. Add 300 µL of PD3 Buffer then mix immediately by inverting the tube 10
   times.
   Centrifuge
   at 16,000 g for 8 minutes. During centrifugation, place a PDH Column in a 2
   mL
   Collection Tube.
5. Transfer all of the supernatant to the PDH Column. Centrifuge at
   14,000-16,000 g
   for 30
   seconds, discard flow-through.
6. Add 600 µL of Wash Buffer into the PDH Column. Centrifuge 14,000-16,000 g
   for 30
   seconds. Discard the flow through then place the PDH Column back in the
   collection tube.
   Centrifuge 14,000-16,000 g for 3 minutes to dry the column matrix. Transfer
   the
   dried
   PDH Column to a new microcentrifuge tube.
7. Add 50 µL of Elution Buffer into the center of the column matrix. Let stand
   for
   at least
   2 minutes to allow elution buffer completely absorbed. Centrifuge
   14,000-16,000
   g for 2
   minutes.

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective and
  safety
  equipment, using aseptic technique, minimizing possibility of producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Gel Purification](#collapseEleven)

###### Gel purification was carried out using the commercial kit from Geneaid Biotech Ltd. with the following instructions explained below.

##### Materials :

###### Equipment

* Microcentrifuge
* Pipettes (1000 µL and 100 µL)
* Scalpel

###### Consumables

* PrestoTM Mini Plasmid Kit
* 1.5 mL microcentrifuge tubes
* Pipette tips (1000 µL and 100 µL)

##### Protocol :

1. Cut the agarose gel slice containing relevant DNA fragments and remove any
   extra
   agarose
   to minimize the size of the gel slice. Transfer up to 300 mg of the gel
   slice to
   a 1.5
   mL microcentrifuge tube.
2. Add 500 µLof Gel/PCR Buffer to the sample then mix by vortex.
3. Incubate at 55-60oC for 10-15 minutes or until the gel slice is
   completely
   dissolved.
4. Place DFH Column in a 2 mL Collection Tube.
5. Transfer 800 µL of the sample mixture to the DFH Column.
6. Centrifuge at 14.000-16.000 g for 30 seconds.
7. Discard the flow-through then place the DFH Column back in the 2 mL
   Collection
   Tube.
8. Add 400 µL of W1 Buffer into the DFH Column.
9. Centrifuge at 14.000-16.000 g for 30 seconds then discard the flow-through.
10. Place the DFH Column back in the 2 mL Collection Tube.
11. Add 600 µL of Wash Buffer into the DFH Column and let stand for 1 minute.
    Centrifuge at
    14.000-16.000 g for 30 seconds then discard the flow-through.
12. Place DFH Column back in the 2 mL Collection Tube.
13. Centrifuge at 14.000-16.000 g for 3 minutes to dry the column matrix.
14. Transfer the dried DFH Column to a new 1.5 microcentrifuge tube. Add 50 µL
    of
    Elution
    Buffer into the center of the column matrix. Let stand for 2 minutes.
15. Centrifuge at 14.000-16.000 g for 2 minutes.

##### Safety Considerations :

* Wearing personal protective safety equipment is necessary
* Keep working area clean and tidy
* Follow necessary handling and disposal considerations by the manufacturer

#### [DNA Restriction](#collapseTwelve)

#### Restriction BsaI

###### Restriction enzyme digestions were performed for restricting BsaI recognition site.

##### Materials :

###### Equipment :

* Pipette
* Cool Box
* Incubator (37oC)
* Heat Block

###### Consumables :

* Nuclease-free water
* CutSmart Reaction Buffer (10X) – NEB
* BsaI
* DNA Sample

##### Protocol :

* The reaction components must be taken in a cool box filled with ice, added
  the
  components in order (water, buffer, DNA and enzymes) to a microcentrifuge
  tube.

|  |  |
| --- | --- |
| Component | 50 µL Reaction |
| DNA | 1 µg |
| 10X CutSmart Buffer | 5 µL (1X) |
| BsaI | 1.0 µL (or 10 units) |
| Nuclease-free water | to 50 µL |

* Mix gently and incubate 1 hour at 37oC.

#### Double Digest EcoRI dan XbaI

###### Restriction enzyme digestions were performed for restricting EcoRI and XbaI recognition site.

##### Materials :

###### Equipment :

* Pipette
* Cool Box
* Incubator (37oC)
* Heat Block

###### Consumables :

* Nuclease-free water
* CutSmart Reaction Buffer (10X) – NEB
* BsaI
* DNA Sample

##### Protocol :

###### Digestion reactions were carried out following the NEB Protocol.

1. The reaction components must be taken in a cool box filled with ice, added
   the
   components in order (water, buffer, DNA and enzymes) to a microcentrifuge
   tube.

|  |  |
| --- | --- |
| Component | 25 µL Reaction |
| DNA | 1 µg |
| 2X TangoTM Buffer | 5 µL |
| EcoRI | 1.0 µL (or 10 units) |
| XbaI | 1.0 µL |
| Nuclease-free water | to 25 µL |

2. Mix gently and incubate 1 hour at 37oC.

##### Safety Considerations :

* Wearing personal protective safety equipment is necessary
* Keep working area clean and tidy
* Follow necessary handling and disposal considerations by the manufacturer

### Parts Characterization

#### [Characterization of BBa\_K381001 on *E.coli* grown in different salinity and nitrate concentrations](#collapseThirteen)

##### Materials :

* LB broth media, LB broth with 25, 35, and 45 ppt salinity and 0, 10, 20
  mM
  nitrate (Potassium nitrate was used) (supplemented with chloramphenicol)
* Plasmid-bearing *E.coli* TOP10 (plasmid containing BBa\_K381001
  part)
* 15 mL centrifuge tubes

##### Protocol :

1. Inoculate Plasmid-bearing *E.coli* TOP10 from solid media to normal
   LB
   broth, LB broth with 25,35, and 45 ppt salinity with variative nitrate
   concentrations (1 oose loop of bacteria to 7 mL broth) and incubate the
   culture
   overnight at 37oC, 200 rpm.
2. After being incubated overnight, check the fluorescence with Glomax by
   inoculating 200 µL culture to 96 well plate and run a fluorescence
   reading.

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective
  and
  safety equipment, using aseptic technique, minimizing possibility of
  producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

#### [Characterization of our new composite part (Bba\_K3252018) on different levels of Vibrio parahaemolyticus supernatant](#collapseFourteen)

##### Materials :

* LB broth media (supplemented with chloramphenicol and ampicillin)
* Sea water LB media
* Wild-type *Vibrio parahaemolyticus*
* Plasmid-bearing *E.coli* TOP10 (plasmid containing BBa\_K3252018
  part)
* 50 mL centrifuge tubes

##### Protocol :

1. Inoculate Plasmid-bearing *E.coli* TOP10 from solid media to normal
   LB
   broth that has been added with ampicillin and cloramphenicol (3 oose
   loops
   of
   bacteria to 15 mL broth) and incubate the culture overnight at
   37oC,
   200 rpm.
2. At the same time, inoculate wild-type *Vibrio parahaemolyticus*
   from
   solid
   media to sea water LB media and incubate the culture overnight at room
   temperature, 150 rpm.
3. After being incubated overnight, centrifuge *V. parahaemolyticus*’
   culture
   to gain its supernatant, and filter the supernatant with 0.22 um
   membrane
   filter.
4. Mix the newly harvested supernatant to fresh LB broth media containing
   ampicillin and chloramphenicol, and make variations of 0%, 10%, 50%
   supernatant-mixed LB broth media.
5. Inoculate overnight culture of plasmids-bearing *E. coli* TOP10 to
   the
   newly made supernatant-mixed LB broth media (150 uL culture to 15 mL
   media).
6. Incubate inoculated culture at 37oC, 200 rpm.
7. Check fluorescence and OD600 of incubated culture every hour
   by
   transferring 100 uL of each culture to transparent 96-well plate and run
   fluorescence and absorbance reading with Glomax Plate Reader by Omega.

##### Safety Considerations :

* Good Microbiological Practice (GMP) is necessary (e.g using protective
  and
  safety equipment, using aseptic technique, minimizing possibility of
  producing
  aerosols)
* After work, all the materials that exposed to cell culture should be
  autoclaved

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# Team:ITB Indonesia/Results

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* Team

  [Team](https://2019.igem.org/Team%3AITB_Indonesia/Team)
  [Attributions](https://2019.igem.org/Team%3AITB_Indonesia/Attributions)
  [Collaborations](https://2019.igem.org/Team%3AITB_Indonesia/Collaborations)
  [Gallery](https://2019.igem.org/Team%3AITB_Indonesia/Gallery)
* Project

  [Description](https://2019.igem.org/Team%3AITB_Indonesia/Description)
  [Design](https://2019.igem.org/Team%3AITB_Indonesia/Design)
  [Experiments](https://2019.igem.org/Team%3AITB_Indonesia/Experiments)
  [Safety](https://2019.igem.org/Team%3AITB_Indonesia/Safety)
  [Model](https://2019.igem.org/Team%3AITB_Indonesia/Model)
  [Hardware](https://2019.igem.org/Team%3AITB_Indonesia/Hardware)
* Parts

  [Parts](https://2019.igem.org/Team%3AITB_Indonesia/Parts)
  [Part Collection](https://2019.igem.org/Team%3AITB_Indonesia/Part_Collection)
* Results

  [Results](https://2019.igem.org/Team%3AITB_Indonesia/Results)
  [Demonstrate](https://2019.igem.org/Team%3AITB_Indonesia/Demonstrate)
* Human

  [Human Practices](https://2019.igem.org/Team%3AITB_Indonesia/Human_Practices)
  [Integrated Human](https://2019.igem.org/Team%3AITB_Indonesia/Integrated_Human)
  [Public Engagement](https://2019.igem.org/Team%3AITB_Indonesia/Public_Engagement)
* Awards

  [Medals](https://2019.igem.org/Team%3AITB_Indonesia/Medals)
  [Awards](https://2019.igem.org/Team%3AITB_Indonesia/Awards)
* [Main Sponsor](https://2019.igem.org/Team%3AITB_Indonesia/MainSponsor)

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#### GOALS

**Shrimpal.id** aim to help local shrimp farmer in Indonesia to prevent major losses caused by shrimp
pathogen, Vibrio parahaemolyticus. This device able to **detect Vibrio parahaemolyticus in relatively low
cell density**, establishing an early warning system for V.parahaemolyticus so further measures can be taken
immediately. Our particular goal is to create a detection device that is **efficient and right on target**.
We also want to **spread biology synthetic** concept, which is not very common in Indonesia, to the public
to raise people’s awareness in biology synthetic as the basic concept of our device.

##### Wet Lab Target:

* **Create and assemble** DNA parts as a circuit of the detection device
* **Characterize** the assembled DNA by **testing** its ability in V.parahaemolyticus **sensing and
  reporting**

##### Modeling target:

* Predict the enzymatic mechanism which involved in the assembled system considering several important
  effecting factors

##### Human practice Target:

* **Implement** technology in a device that solve problems in shrimp ponds efficiently
* **Disseminate** biology synthetic concepts as broad as possible to various elements in the society

#### RESULTS

We started to build Shrimpal.id step by step. Once the project was finished, the conclusions reached at this
following page:

##### Wet Lab

* ###### We have built a complete Part Collection of standardized Golden Gate and BioBricks basic parts. It is a fully functional DNA collection with their characterization of this parts.
* ###### We have purified and 16s rDNA characterized Vibrio parahaemolyticus that we used in our project. V. parahaemolyticus that we used was obtain from local isolate in Balai Perikanan Budidaya Air Payau Situbondo, Indonesia.
* ###### We already made V. parahaemolyticus standard curve to determine cell number based OD600

This standard curve used to convert the OD600 data of V.parahaemolyticus to its estimated cell
numbers

![](https://static.igem.org/mediawiki/2019/5/51/T--ITB_Indonesia--results1.png)

Figure 1. V.parahaemolyticus standard curve

This standard curve used to convert the OD600 data of *V. parahaemolyticus* to its
estimated cell numbers.
OD600 values were measured using UV-Vis Spectrophotometer, while cell numbers were
estimated
using total
plate count method. The OD600 values used are within the range of linearity (0,2-0,7) and
the
r2 value
shows a good correlation between both variables. The equation obtained from the curve is shown
below.

OD600 = (cell number) x 10-9 + 0,1232

* ###### We have characterize BBa\_K381001 parts on Escherichia coli grown in different salinity and nitrate concentrations.

Nitrate or nitrite has a negative correlation to the growth of Vibrio paahaemolyticus. The dynamics of ammonium, nitrate, and nitrite concentrations are complex, and so it can be difficult to interpret the multivariate associations of thesespatially - and temporally -indexed variables. Overall though, positive associations with nutrients indicates that availability of nitrogen and phosphorous provide a hospitable environment for the bacterium. Here we tried to characterize nitrate sensor ability of part BBa\_K381001 that transformed into E.coli
TOP10 grown in different level of salinity and here is the result :

![](https://static.igem.org/mediawiki/2019/0/07/T--ITB_Indonesia--results2.png)

Figure 2.Bar graphic of nitrate sensing in a range of salinity. The data shows that in
a higher salinity (>10 ppt), nitrate sensing ability of the culture decrease since the higher
nitrate concentration doesn’t result in higher fluorescence.

In this project we use nitrate reporter consists of sensitive promoter PyeaR with a GFP coding device
and strong RBS to create nitrate-sensitive system which signal through GFP. In the normal condition,
the promoter PyeaR is repressed by NsrR, a protein native to E.coli cells. But, when nitrate or
nitrite enter the cell, it is converted to nitric oxide halting the repression and allowing the
production of GFP.

In the previous characterization done by BCCS Bristol in iGEM 2010, this part can be used to detect
nitrate in concentration range 2-100 mM. We previously predict that between this range of nitrate
concentration this nitrate sensor could work well.

![](https://static.igem.org/mediawiki/2019/9/96/T--ITB_Indonesia--results3.png)

Figure 3. Nitrate sensing report of transformant E.coli grown in Normal-salinity LB
(10 ppt)

Figure 3 shows that in a media with 10 ppt salinity, the correlation between nitrate concentration
and fluorescence is considered linear (R2=0.916). In a higher nitrate concentration, the
fluorescence level also raised.

Unfortunately the same correlation can’t be accomplish in salinity level higher than usual medium
condition (>10 ppt). We predict that this phenomena probably caused by disruption of cell growth in
a higher salt concentration. It supported by the growth curve data of E.coli grown in various media
with different salinity as shown in figure 4.

![](https://static.igem.org/mediawiki/2019/d/d2/T--ITB_Indonesia--results4.png)

Figure 4. E.coli TOP10 growth curve in various salinity. Higher salinity produce
relatively lower cell density

We can see that the higher salinity level, the lower cell growth. Culture grown in 25ppt LB grew a
little lower than normal LB, while the variation with 35 and 45 ppt salinity shows even lower
growth. Culture grown in sea water LB probably produce a higher growth because sea water might
contains lots of supporting materials for cell growth.

However, E.coli that we used still managed to grow even in a relatively higher salinity. This
behaviour might caused by adaptation mechanism of cell in stress condition. One of the possible
mechanism is hyperosmotic response. Hyperosmotic condition causes water loss from cytoplasm. E. coli
responds to osmotic shock by increasing K+ ion influx through Trk, Kdp, and Kup uptake systems. The
cell also excretes putrescine (a divalent cationic molecule), to balance out intracellular ionic
strength. Glutamate synthesis follows to further balance the intracellular ionic strength. At this
point, the cells’ turgor pressure are restored. The role of K+/glutamate is replaced by trehalose, a
nonionic compatible solute. K+ and glutamate levels drop as trehalose level increases. Normal cell
functions are restored after trehalose accumulation occurs (Moat, et al,2003).

This salinity problem actually could be handled by transforming salinity resistant plasmid into the
cell, for example part BBa\_K729005. This part produce IrrE protein originates from Deinococcus
radiodurans that upregulate the production of several stress-responsive protein, protein kinase,
metabolic protein, and detoxification protein, but we didn’t use this method because our design
already used two relatively large plasmid. Transforming this other plasmid could possibly aggravate
the cell’s metabolic burden.

* ###### We have characterize full system integration of our composite part BBa\_K3252044 on different levels of Vibrio parahaemolyticus filtered supernatant

Here we tried to characterize our new composite part

![](https://static.igem.org/mediawiki/2019/4/43/T--ITB_Indonesia--results5.png)

Figure 5 GFP/OD600 level in different supernatant concentrations. Higher supernataht
concentration produces higher level of GFP/OD600 readings

From figure above (Fig. 5), it can be seen that the GFP/OD600 level positively correlates with the
concentration of supernatant in the medium. This pattern starts to be observable after 1 hour of
incubation, while at the initial incubation time (hour=0) there is no conclusive pattern observed.
However, GFP/OD600 read after the first hour of incubation decreased steadily until the end of our
observation time (3 hours).This is caused by the breakdown of the signal molecule, autoinducer (AI) While it is highly stable in low pH, its half-life is reduced to <3 hours in slightly alkaline condition (Leadbetter and Greenberg, 2000). The pH of salt water itself ranges from 7,5-8,5. The sample added contains no viable V. parahaemolyticus cells, hence no additional AI is produced during incubation period.

We are very proud of this finding as this shows that our system works as expected. The supernatant of
Vibrio parahaemolyticus is supposed to be containing its very own autoinducer, which would be
recognized by our constructed part LuxN, and induced the latter cascading process which ends up
expressing green fluorescence protein. The increased GFP level along with increased supernatant
level means the system works in a well manner.

These findings also indicate that the best formula for the observation is 50% filtered Vibrio
parahaemolyticus supernatant, while the best time to observe the Vibrio parahaemolyticus level is
after one hour of incubation. This insight would later be very meaningful in the development of the
project.

#### HUMAN PRACTICE

WUntil now, we have achieved impressive human practices events that we held. See [our page](https://2019.igem.org/Team%3AITB_Indonesia/Human_Practices) for detailed
information.

#### CONSIDERATIONS FOR REPLICATING THE PROJECT

* Ligation reaction for parts assembly need to be optimized
* **Parts composite stability**: We are using two relatively large plasmids transformed into one cell that
  technically not easy to maintain. We recommend to increase antibiotic dosage and perform routine
  subculture, or using transformant cell from glycerol stock for each experiment
* To asses composite part’s ability in V.parahaemolyticus sensing and reporting, make sure the
  V.parahaemolyticus supernatant already filtered
* In PCR colony reactions to check the gene transformation, make sure the cells are diluted first in
  NFW and not too cloudy nor too clear
* Better use an automatic sampler and absorbance/fluorescence reader to check the culture, if possible

#### FUTURE PLANS

We have successfully constructed and cloned part composite that build our sensor device. Our future aim
is to determine and increase the sensitivity of detection. We also want to evaluate biosafety and
biosecurity issues that might be encountered in the actual usage.

Moreover, hopefully in the future we could design and develop integrated circuit to process the data and
the software that will deliver V.parahaemolyticus level to the shrimp farmer and give suggestion on
further measures

#### REFERENCE

* Leadbetter, Jared R. and EP Greenberg. 2000. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. Journal of Bacteriology. 182 (24): 6921-26
* Moat, A. G., Foster, J. W., & Spector, M. P. (Eds.). (2003). Microbial physiology. John Wiley &
  Sons. P.583-584

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