iGEM 2017 — Team NTNU_Trondheim

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# Team:NTNU Trondheim/Notebook

* [Home](https://2017.igem.org/Team%3ANTNU_Trondheim)
* [Safety](https://2017.igem.org/Team%3ANTNU_Trondheim/Safety)
* [Parts](https://2017.igem.org/Team%3ANTNU_Trondheim/Parts)
* Project

  + [Description](https://2017.igem.org/Team%3ANTNU_Trondheim/Description)
  + [Background](https://2017.igem.org/Team%3ANTNU_Trondheim/Background)
  + [Results](https://2017.igem.org/Team%3ANTNU_Trondheim/Results)
  + [Contribution](https://2017.igem.org/Team%3ANTNU_Trondheim/Contribution)
  + [Demonstrate](https://2017.igem.org/Team%3ANTNU_Trondheim/Demonstrate)
  + [Modeling](https://2017.igem.org/Team%3ANTNU_Trondheim/Model)
  + [Lab Journal](https://2017.igem.org/Team%3ANTNU_Trondheim/Notebook)
  + [Protocols](https://2017.igem.org/Team%3ANTNU_Trondheim/Experiments)
* Human practices

  + [Human practice](https://2017.igem.org/Team%3ANTNU_Trondheim/HP/Silver)
  + [Public engagement](https://2017.igem.org/Team%3ANTNU_Trondheim/Engagement)
* People

  + [Team](https://2017.igem.org/Team%3ANTNU_Trondheim/Team)
  + [Collaborations](https://2017.igem.org/Team%3ANTNU_Trondheim/Collaborations)
  + [Attributions](https://2017.igem.org/Team%3ANTNU_Trondheim/Attributions)
  + [Sponsors](https://2017.igem.org/Team%3ANTNU_Trondheim/People/Sponsors)
* Awards

  + [Achievements](https://2017.igem.org/Team%3ANTNU_Trondheim/Achievements)
  + [Applied Design](https://2017.igem.org/Team%3ANTNU_Trondheim/Applied_Design)
  + [Hardware](https://2017.igem.org/Team%3ANTNU_Trondheim/Hardware)

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# Lab Journal

Below follows our lab journal giving a daily description of work done on and in association with the lab.
This includes both standard gene-manipulation, growth of bacteria, hardware setup, 3D-modelling and printing.

To download the document [click here](https://static.igem.org/mediawiki/2017/a/a3/T--NTNU_Trondheim--Lab_journal.pdf).

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Email: igem-team@ntnu.no

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# Team:NTNU Trondheim/Experiments

* [Home](https://2017.igem.org/Team%3ANTNU_Trondheim)
* [Safety](https://2017.igem.org/Team%3ANTNU_Trondheim/Safety)
* [Parts](https://2017.igem.org/Team%3ANTNU_Trondheim/Parts)
* Project

  + [Description](https://2017.igem.org/Team%3ANTNU_Trondheim/Description)
  + [Background](https://2017.igem.org/Team%3ANTNU_Trondheim/Background)
  + [Results](https://2017.igem.org/Team%3ANTNU_Trondheim/Results)
  + [Contribution](https://2017.igem.org/Team%3ANTNU_Trondheim/Contribution)
  + [Demonstrate](https://2017.igem.org/Team%3ANTNU_Trondheim/Demonstrate)
  + [Modeling](https://2017.igem.org/Team%3ANTNU_Trondheim/Model)
  + [Lab Journal](https://2017.igem.org/Team%3ANTNU_Trondheim/Notebook)
  + [Protocols](https://2017.igem.org/Team%3ANTNU_Trondheim/Experiments)
* Human practices

  + [Human practice](https://2017.igem.org/Team%3ANTNU_Trondheim/HP/Silver)
  + [Public engagement](https://2017.igem.org/Team%3ANTNU_Trondheim/Engagement)
* People

  + [Team](https://2017.igem.org/Team%3ANTNU_Trondheim/Team)
  + [Collaborations](https://2017.igem.org/Team%3ANTNU_Trondheim/Collaborations)
  + [Attributions](https://2017.igem.org/Team%3ANTNU_Trondheim/Attributions)
  + [Sponsors](https://2017.igem.org/Team%3ANTNU_Trondheim/People/Sponsors)
* Awards

  + [Achievements](https://2017.igem.org/Team%3ANTNU_Trondheim/Achievements)
  + [Applied Design](https://2017.igem.org/Team%3ANTNU_Trondheim/Applied_Design)
  + [Hardware](https://2017.igem.org/Team%3ANTNU_Trondheim/Hardware)

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# Protocols

Throughout the project we used and developed several protocols for creation of culture media and
conduction of experimental procedures. Among these were phage isolation, creation of supercompetent cells,
transformation, DNA purification, restriction digestion, gel electrophoresis, TECAN analysis and PCR.
The procedures where conducted as described below unless otherwise described in
[the lab journal](https://2017.igem.org/Team%3ANTNU_Trondheim/Notebook).

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# Media

All media should be mixed using a magnetic stirrer in a bottle twice as large as the liquid volume,
and autoclaved at 121 °C for 20 minutes.

**Luria-Bertani broth (LB)**
· Reverse osmosis water (1 L)
· Bacto tryptone (10 g)
· Yeast extract (5 g)
· NaCl (10 g)

**Luria-Bertani agar (LA)**
· LB medium (1 L)
· Agar (15 g)

**Phosphate-buffered saline (PBS)**
· Distilled water (1 L)
· NaCl (8 g)
· KCl (0.2 g)
· Na2HPO4 (1.42 g)
· KH2PO4 (0.24 g)

NB: If you are making agar plates, add 15 g agar to the solution before autoclaving. Add the appropriate
antibiotic to the agar solution after cooling the solution to 50 °C.

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# Antibiotics

Work in a fume hood when preparing antibiotic stock solutions. For each solution, dissolve the
antibiotic in water or ethanol, and sterile filter through a 0.2 μm filter. Aliquot into 1.5 mL
Eppendorf tubes and store in the freezer at -20 °C.

**Kanamycin (50 mg/mL stock solution)**
· Kanamycin (250 mg)
· Reverse osmosis water (5 mL)

**Ampicillin (50 mg/mL stock solution)**
· Ampicillin (250 g)
· Reverse osmosis water (5 mL)

**Chloramphenicol (35 mg/mL stock solution)**
· Chloramphenicol (175 mg)
· 96% ethanol (5 mL)

**Spectinomycin (100 mg/mL stock solution)**
· Spectinomycin (500 mg)
· Reverse osmosis water (5 mL)

![](https://static.igem.org/mediawiki/2017/5/52/T--NTNU_Trondheim--splitter_2.svg)

# Experiments

Throughout the project we used and developed several protocols for experimental procedures. Among these were phage isolation, creation of supercompetent
cells, transformation, DNA purification, restriction digestion, gel electrophoresis, TECAN analysis
and PCR. The procedures where conducted as described below unless otherwise described in
[the lab journal](https://2017.igem.org/Team%3ANTNU_Trondheim/Notebook).

Show more

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# Isolation of bacteriophages

**Materials**
· Overnight culture of E. coli DH5a
· 125 mL Erlenmeyer flask
· 40 mL waste water
· LB medium
· Warm LB agar plates
· Warm top agarose (0.7 %)
· 37 °C incubator with shaker platform
· 37 °C incubator
· 45 °C water bath

**1. Amplification of bacterophage**

* Pipette 5 mL LB medium into flask containing 40 mL of waste water
* Inoculate the waste water in the flask with 5 mL of overnight culture of E. coli
* Incubate culture at 37 °C, shaking for 24 h

Prior to session 2, 2.5 mL overnight E. coli culture was inoculated with 25 mL LB-medium, and incubated at 37 C shaking until OD600 = 0.8.

**2. Bacteriophage isolation and plating**

* Transfer 10 mL of waste water-bacteria-phage culture into a centrifuge tube, and centrifuge the sample at 2000 rpm for 5 min.
* Pipette the supernatant into a 10 mL syringe barrel fitted with 0.2 micron filter. Storage tube with phages can be stored at 4 °C for several months. Vortex this tube before serial dilutions.
* Prepare a series of microcentrifuge tubes for serial 10-fold dilutions. Label 10 tubes 1-10. In each tube, pipette 0.9 mL of sterile LB.
* Serial dilution: Transfer 0.1 mL of phage suspension into tube 1, and vortex. Using the same pipette, transfer 0.1 mL of the sample from tube 1 into tube 2, and vortex. Repeat this process, transferring 0.1 mL from tube 2 to tube 3, and so on, mixing each time.
* Get ready 0.7 % top agarose in a falcon tube and let it be "cooled" to 45 °C in water bath. (Turn on water bath and wait until 45 °C)
* Label 11 new small falcon tubes with the number 1-10 ("1-10" phage+DH5a and "Control" containing only DH5a). Distribute 0.5 mL of log-phase E. coli in each of the small falcon tubes.
* To each tube of bacteria, add 0.1 mL of corresponding phage dilution. Note: if you work from the most diluted to the least, you can use the same pipette. Mix gently by inverting (NO VORTEX).
* Incubate at 37 °C for 10 min.
  * In the meantime, label 11 warm, agar plates. Keep the plates in the 37 °C incubator until you are ready to use them.
  * Collect pre-warmed plates from incubator. Pipette 3 mL of top agarose to the cell-phage tube. Cap the tube, and mix gently inverting it 3 times. Quickly pour the mixture onto warmed plate. You can tip the plate slightly to spread the top agarose. Push plate aside, but do not pick it up until agarose solidifies.
  * Air dry with open lid for 15 min in the sterile hood.
  * When the top agarose has solidified, incubate the plates, inverted at 37 °C for 3 h.

**3. Examiniation of bacteriophage plates, phage storage**

* Count plaques on plates (See lab journal day 22)
* Pick an isolated plaque for long-term storage: pipette 1 mL of LB into a small Eppendorf tube, and add 1 drop of chloroform. Use a small pipette tip and pierce the agar surrounding plaque, and pick out the agar containing plaque. Place the agar with plaque into 1 mL of LB. Store the plaque in refrigerator (4 °C).

# Supercompetent cell production

**Day 1**

1. Make an E. coli DH5α culture in a small flask with 10 mL Psi-medium. Inoculate culture at the end of the day (4 pm), leave in 37 °C shaking incubator overnight.

2. Prepare transformation buffers (TFB) for day 2.

TFB 1 (200 mL)

* Add the following to a 250 mL flask:
○ Potassium acetate (0.588 g)
○ Rubidium chloride (2.42 g)
○ Manganese chloride (2.00 g)
○ Glycerol (30 mL)* Fill up with MilliQ water to a total volume of 180 mL
* Adjust pH to 5.8 with a pH meter by adding diluted acetic acid (0.2 M)
* Fill up with MilliQ water to a total volume of 200 mL

TFB 2 (100 mL)

* Add the following to a 250 mL flask:
○ MOPS (0.21 g)
○ Rubidium chloride (0.121 g)
○ Calcium chloride (1.1 g)
○ Glycerol (15 mL)* Fill up with MilliQ water to a total volume of 90 mL
* Adjust pH to 6.5 with a pH meter by adding diluted NaOH (0.2 M)
* Fill up with MilliQ water to a total volume of 100 mL

**Day 2**

1. Start culture as early as possible. Transfer 1 mL culture to a new shaking flask with 100 mL Psi-medium. Grow in shaking incubator at 37 °C until OD600 reaches 0.4.
2. Place the culture on ice for 15 minutes, along with TFB1 and TFB2.
3. Centrifuge the culture for 5 minutes at 4000 rpm at 4 °C. Pour off supernatant. (Safe break point, if you leave cells on ice.)
4. Resuspend pellet carefully in 40 mL TFB1. Let sit for 5 minutes on ice.
5. Centrifuge for 5 minutes at 4000 rpm at 4 °C. Pour off supernatant. Resuspend pellet carefully in 3 mL TFB2.
6. Mark as many 1.5 mL Eppendorf tubes as you will need with cell strain name and the date.
7. Aliquot 100 uL of resuspended cells into the 1.5 mL Cryo tubes. Leave all tubes on ice until finished aliquoting. Flash freeze for 5-10 seconds in liquid nitrogen, store at -80 °C.

# Transformation

1. Thaw supercompetent cells on ice for approximately 10 minutes.
2. Using a pipette, transfer 1 μL of mini-prepped plasmid into the cells. Pipette up and down while stirring the pipette tip gently around the tube for 5-10 seconds.
3. Incubate the cells on ice for 20 minutes (can be shortened to 5 if you are in a hurry).
4. Heat shock the cells for 45 seconds at 42 °C. Immediately put cells on ice for 2 minutes after.
5. Add 900 uL LB medium to the cells. Incubate at 37 °C, 200 RPM for 1 hour (can be shortened to 45 minutes if you are in a hurry).
6. Plate out 100 uL of the transformation mixture onto agar plates with the appropriate selection agent

# DNA purification (MiniPrep)

1. Centrifuge 1 mL of bacterial culture in a clear 1.5 mL tube at full speed (16,000 g) for 20 seconds in a microcentrifuge. Discard supernatant.
2. Add 200 μL of P1 buffer (red) to the tube and resuspend the pellet completely by pipetting.
3. Add 200 μL of P2 buffer (green), mix by inverting the tube 2-4 times, and incubate for 1-2 minutes. Do not vortex.
4. Add 400 μL of P3 buffer (yellow) and mix gently but thoroughly. Allow the lysate to incubate at room temperature for 1.2 minutes. Do not vortex.
5. Centrifuge samples for 4 minutes at 16,000 g.
6. Plave a Zymo-Spin IIN column in a collection tube and transfer the supernatant from step 5 into the column. Avoid distrurbing the pellet.
7. Centrifyge the Zymo-Spin IIN assembly for 30 seconds at 16,000 g.
8. Discard the flow-through and retyrn the column to the same collection tube.
9. Add 200 μL of Endo-Wash Buffer to the column and centrifuge for 30 seconds at 16,000 g.
10. Add 400 μL of Plasmid Wash Buffer to the column and centrifuge for 1 minute at 16,000 g.
11. Transfer the column into a clean 1.5 microcentrifuge tube and then add 30 uL of DNA Elution Buffer directly to the column matrix and incubate for one minute at room temperature.
12. Centrifuge for 30 seconds at 16,000 g to elute the plasmid DNA.

Store the plasmid at -20 degrees.

# Restriction digestion of DNA

**Materials**

* Up to 1 μg DNA (0.5 ug)
* Appropriate buffer (2 μL)
* Restriction enzyme (0.5-1 μL)
* Distilled water

**Method**

1. Mix buffer and DNA, and fill up to 20 μL with distilled water. Mix well.
2. Add enzyme, pipette up and down with a pipette several times to mix.
3. Incubate for 2 hours or overnight at 37 ˚C in a heat block.

NB! Enzymes should always be kept in an ice rack when outside of the freezer. Always wear gloves when handling enzymes.

# Gel electrophoresis

1. Pour agarose gel (recipe below) containing the appropriate\* DNA binding dye into a gel tray of appropriate size. Make sure the tray is tightly put into the gel tub.
2. After the gel has set, add TAE buffer until the gel is completely submerged in buffer.
3. Add DNA ladder with dye to at least one well in the gel.
4. Add your DNA samples to be separated into separate wells.
5. Connect the gel tub to a power supply, and run the gel at 80-110 V until the color bands are approximately 75 % to 80 % down the length of the gel.
6. Disable the power supply and put the gel into the imaging apparatus.
7. On the computer open the ImageLab program.
8. Select the appropriate\* protocol, position gel correctly and take image.
9. Print image and save the image file to the iGEM2016 folder (C://). Use a descriptive name for the file.
10. If running gels to excise DNA fragments, attach the UV screen to the drawer.
11. Put on orange safety goggles.
12. Turn on light (top button)
13. Excise the desired bands using a scalpel. Clean the scalpel before and between excisions. Transfer the excised gel bands to clean 1.5 mL Eppendorf tubes, and label them clearly. Store in freezer.
14. Dispose of the remaining gel in the blue barrel on the floor next to the imaging apparatus.
15. Rinse gel tray and gel tub in water and leave to dry.

\*If excising bands use GelGreen. Otherwise use GelRed.

Always use gloves and safety goggles when working with gels.

**Agarose gel recipe (0.8 %):**

1. Add TAE buffer (400 mL) to 400 mL glass bottle
2. Add agarose (3.2 g) to the buffer
3. Boil in the microwave oven for approximately 5 min at medium power
4. Add GelRed or GelGreen (20 μL) (gelGreen if you want to cut out the band)

**Loading of samples**

* 2 μL ladder
* 20 μL sample + 4 μL loading dye (mixed)
* Run for 45 minutes
* Take picture in the ChemiDoc and print out

# TECAN infinite M200 PRO

Two scripts are utilized for OD-measurements.

**Script No.1**

* Hold Temp: 37˚C
* Performed: N-cycles
* Each cycle is measured at absorbance 600 nm in 300 uL culture, 25 flashes per reading
* Shaking:
* Duration: 300 second
* Mode: Orbital
* Amplitude: 2.5 mm
* Frequency: 244 rpm

**Script No.2**

* Hold Temp: T
* Performed: 24-cycles
* Fluorescence intensity scan with settings
* Scan selection: Emission scan
* Mode: Top (from top)
* Gain: manual = 255

Integration

* Lag time: 100 μSec
* Integration time: 20 uSec
* 25 flashes per reading
* Excitation wavelength: 390 nm
* Emission wavelength: 450-540 nm
* Step size: 10 nm
* Z-position: 18000 μm

Shaking:

* Duration: 300 second
* Mode: Orbital
* Amplitude: 2.5 mm
* Frequency: 244 rpm

# PCR (Polymerase chain reaction)

Kit: EHIFI-RO Roche "Expand High Fidelity PCR System"
Procedure: The general instruction-sheet has not been followed.
Changed made are described below:

1. Briefly vortex and centrifuge all reagents before starting.
2. Prepare two mixes in a sterile microfuge tubes (on ice):

**Mix 1 (for one reaction)**

|  |  |
| --- | --- |
| **Reagent** | **Amount** |
| Sterile double-dist. Water | 8 μL |
| Deoxynucleotide mix, 10 mM of each dNTP) | 1 μL |
| Upstream primer | 2 ng/L |
| Downstream primer | 2ng/L |
| Template DNA | 1 μL, 20 ng |

**Mix 2 (for one reaction)**

|  |  |
| --- | --- |
| **Reagent** | **Amount** |
| Sterile double-dist. Water | 18 μL |
| Expand High Fidelity buffer, 10×conc. with 15 mM MgCl2 | 5 μL |
| Expand High Fidelity enzyme mix (polymerase) | 0.75 μL |
| dMSO | 1.25 μL |

Combine Mix 1 and Mix 2 in a thin-walled PCR tube (on ice).
Gently vortex the mixture to produce a homogeneous reaction, then centrifuge briefly to collect sample at the bottom of the tube.

# DNA assembly (NEBuilder)

The mass of each fragment can be measured using NanoDrop. Recommended amount of fragments used for assembly:

|  |  |  |  |
| --- | --- | --- | --- |
|  | **2-3 fragment assembly\*** | **4-6 fragment assembly\*\*** | **Positive control** |
| **DNA ratio (vector : insert)** | 1 : 2 | 1 : 1 | - |
| **Total amount of fragments** | X μL | X μL | 10 μL |
| **DNA Assembly Master Mix** | 10 μL | 10 μL | 10 μL |
| **Deionized water (dH2O)** | 10-X μL | 10-X μL | - |
| **Total volume** | 20 μL | 20 μL | 20 μL |

\*Optimized cloning efficiency is 50-100 ng of vectors with 2 fold excess of inserts. Use 5 times more inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
\*\*To achieve optimal assembly efficiency, it is recommended to design >20 bp overlap regions between each fragment with equimolarity.

1. Thaw and vortex master mix thoroughly and keep on ice.
2. Set up the following reaction (see table) in a PCR tube on ice:
   vector (backbone) + insert (plasmid) + dH2O + master mix
3. Incubate samples in a thermocycler at 50 C for 15 minutes (2-3 fragments) or 60 minutes (4-6 fragments).
4. Store samples on ice or at -20 C for subsequent transformation.

# MP6 reversion assay

In order to characterize our MP6 biobrick's mutational properties and compare them to those
of the original MP6, we used the following protocol:

1. Doubletransform E.coli with MP6 and pla230 (Addgene reversion assay plasmid) and let it incubate for 18 hours exactly.
2. Split the culture and add glucose to a final concentration of 20 mM to one subculture, the same concentration of arabinose to the other.
3. Incubate for 24 hours.
4. Plate on LA+Amp+glucose and LA+glucose plates in 10x dilutions.
5. Count after the colonies have grown sufficiently

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# Team:NTNU Trondheim/Results

* [Home](https://2017.igem.org/Team%3ANTNU_Trondheim)
* [Safety](https://2017.igem.org/Team%3ANTNU_Trondheim/Safety)
* [Parts](https://2017.igem.org/Team%3ANTNU_Trondheim/Parts)
* Project

  + [Description](https://2017.igem.org/Team%3ANTNU_Trondheim/Description)
  + [Background](https://2017.igem.org/Team%3ANTNU_Trondheim/Background)
  + [Results](https://2017.igem.org/Team%3ANTNU_Trondheim/Results)
  + [Contribution](https://2017.igem.org/Team%3ANTNU_Trondheim/Contribution)
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  + [Hardware](https://2017.igem.org/Team%3ANTNU_Trondheim/Hardware)

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# Results

Here is a review of our results. Both for the phage evolution project, our new biobrick
and the characterization of an already existing part; the lux operon.

![](https://static.igem.org/mediawiki/2017/5/52/T--NTNU_Trondheim--splitter_2.svg)

# Phage Evolution

For phage evolution we first characterized the phage that we purified from sewage samples and compared
it to T2 phage that we had in the lab. Our phage solution was found to be significantly more effective
against our E.coli strain. For future experiments we also tested how much the growth phase of the host
bacterium would affect phage infection efficiency. Phage showed clear infection curves at all initial
bacterial ODs from 0.3 to 0.9. Interestingly at higher OD the curve became biphasic. The time for the
first lysis to take place varied, increasing with initial OD, but stayed within 1 and 2 hours after
induction.

![](https://static.igem.org/mediawiki/2017/a/a8/T--NTNU_Trondheim--Results_01.png)

Comparing the phage we extracted from sewage (No. 2) with the T2 phage from the lab (No.1). The curve shows OD600 of a 300 μL E.coli culture induced with phage over time after induction.

![](https://static.igem.org/mediawiki/2017/4/49/T--NTNU_Trondheim--Results_02.png)

*Escherichia coli* cultures grown to different ODs challenged with phage from the sewage.

Having characterized our own phage, we evolved a resistant bacterium against it using a single chemostat
setup over the course of 24 hours. Samples of the culture were then plated out and three resistant bacterial
colonies were further studied (Res1 , 2 and 3). We characterized them together with the unevolved E.coli
strain (control) for resistance to phage. We found that none of the resistant colonies showed signs of
infection after two hours, but after five hours one of the two repeats of Res2 and both repeats of Res3
were lysed, indicating that phage that were potent against these strains were present in small quantities
in our sewage phage sample. Five hours is about three generations. Thus the occurrence frequency of these
phage in the original phage sample was about 10-6-10-7.

![](https://static.igem.org/mediawiki/2017/7/77/T--NTNU_Trondheim--Results_03.png)

Three strains of phage-resistant *E.coli* challenged with phage they were evolved against (sewage phage).

We then performed a round of phage evolution using our three chemostat system on the original *Escherichia coli*
strain and the Res1 strain (that was not lysed by the original phage even after 8 hours). We purified
phage from the phage-evolving chemostat and tested the phage on our original strain as well as on all the
resistant ones. Our new phage sample lysed all strains within 1.5 hours after induction, giving us the
proof of concept that we were aiming for. That is, we first evolved bacteria that were resistant to our
original phage strain and then we managed to evolve a new strain of phage that could lyse the resistant
bacteria!

![](https://static.igem.org/mediawiki/2017/d/d8/T--NTNU_Trondheim--Results_04.png)

Three strains of phage-resistant *E.coli* challenged with phage evolved in chemostat.

![](https://static.igem.org/mediawiki/2017/6/6d/T--NTNU_Trondheim--splitter_1.svg)

# Biobrick characterization

The MP6 mutator (original) biobrick was derived from the MP6 original plasmid, and it is an arabinose
inducible and glucose repressible plasmid that codes for 6 mutagesis related genes
(see [MP6 Part site](http://parts.igem.org/Part%3ABBa_K2265000) for details).

To test its mutagenic activity after inducer and repressor addition, we used a reversion assay based on
the plasmid pLA230 (from Addgene). The pLA230 plasmid contains a non-functional Ampicillin resistance gene.
The inactivity of the gene is due to a point mutation, and a reversion of this mutation will lead to
Ampicillin resistance in a cell containing the plasmid.

In order to characterize the mutation rate of the MP6 biobrick, it was transformed together with
pLA230 into E.coli DH5α. The doubly transformed cells were grown with either glucose or arabinose
(see [protocol](https://2017.igem.org/Team%3ANTNU_Trondheim/Experiments)) and incubated in 37ºC overnight. The cells were then plated on agar containing Ampicillin.
The number of colonies formed was an indication of the mutation activity of MP6, as only bacteria containing
pLA230 where the point mutation was reverted would survive on the Amp containing agar plates.

![](https://static.igem.org/mediawiki/2017/d/d5/T--NTNU_Trondheim--Results_05.png)

Reversion frequency of MP6 biobrick induced with arabinose or repressed with glucose.

The reversion frequency was quantified by dividing the number of colonies surviving on ampicillin by
the number of colonies growing without ampicillin (scaling with the dilution factors accordingly).
We performed two testing rounds on our MP6 biobrick, giving on average a reversion frequency of 10-5
when induced and 10-6 when repressed. In order to compare our biobrick with the original MP6 we
quantified the relative reversion frequencies for both, that is how many times more revertants we
got when the plasmid was induced than when it was not. We got ratios of 19 and 21 respectively.
Thus the two plasmids performed quite similarly.

![](https://static.igem.org/mediawiki/2017/3/3c/T--NTNU_Trondheim--Results_06.png)

Relative reversion frequencies of our MP6 biobrick and orginal MP6.

![](https://static.igem.org/mediawiki/2017/5/52/T--NTNU_Trondheim--splitter_2.svg)

# Lux characterization

We also imporved the characterisation of the biobrick [BBa\_K325909](http://parts.igem.org/Part%3ABBa_K325909),
which is an operon consisting of
Lux C, D, A, B, E under the arabinose-induced promoter pBAD. We chose to improve this biobrick as we
originally wanted to use the lux operon as a reporter for our arabinose-inducible system. In this way,
we could test how the expression from the pBAD promoter was dependent on arabinose concentration.

By looking at previous characterisation results of the biobrick, we thought it would work reasonably
to use DH5α competent *E. coli* cells and an incubation temperature of 37 °C. However, when testing with
0.01M arabinose concentrations, no observable luminescence was detected at any time after induction.
Therefore, we decided to characterise the biobrick by measuring the luminescence from the lux operon
at different arabinose concentrations and temperatures. We measured the luminescence with a Tecan M200
plate reader, using four different temperatures and six different arabinose concentrations. As the
machine could only perform heating and not cooling, it was challenging to measure at temperatures
below 29 °C.

Results from our experiments are shown in the graphs below. Error bars are averages over 3 repeats.
As no luminescence was detected at 37 °C, these graphs are not included. The optimal temperature for
Lux luminescence as determined by us was 28.8 °C.

![](https://static.igem.org/mediawiki/2017/a/a8/T--NTNU_Trondheim--Results_07.png)

Lux luminescence at 24.0 °C. The developement is slow and the values are low.

![](https://static.igem.org/mediawiki/2017/6/69/T--NTNU_Trondheim--Results_08.png)

Lux luminescence at 28.8 °C. This is the optimal temperature for luminescence as measured by us.

![](https://static.igem.org/mediawiki/2017/9/92/T--NTNU_Trondheim--Results_09.png)

Lux luminescence at 29.3 °C. At 1e-5 M concentration luminescence is no longer detected.

![](https://static.igem.org/mediawiki/2017/e/ea/T--NTNU_Trondheim--Results_10.png)

Lux luminescence at 33.4 °C. Rather slow and poor performance at all inducer concentrations.

![](https://static.igem.org/mediawiki/2017/7/78/T--NTNU_Trondheim--Results_11.png)

The average luminescence over 4.5 hours at 1e-2 M Arabinose concentration against incubation temperature.

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