iGEM 2019 — Team Groningen

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# Team:Groningen/Notebook

iGEM Groningen 2019 :: Notebook

![iGEM Groningen](https://static.igem.org/mediawiki/2019/e/eb/T--Groningen--logo_transparent.png)

* [Home (current)](https://2019.igem.org/Team%3AGroningen)
* Team
  + [Team Members](https://2019.igem.org/Team%3AGroningen/Team)
  + [Collaborations](https://2019.igem.org/Team%3AGroningen/Collaborations)
  + [Sponsors](https://2019.igem.org/Team%3AGroningen/Sponsors)
  + [Attributions](https://2019.igem.org/Team%3AGroningen/Attributions)
* Project
  + [Description](https://2019.igem.org/Team%3AGroningen/Description)
  + [Design](https://2019.igem.org/Team%3AGroningen/Design)
  + [Experiments](https://2019.igem.org/Team%3AGroningen/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AGroningen/Notebook)
  + [Model](https://2019.igem.org/Team%3AGroningen/Model)
* Impact
  + [Results](https://2019.igem.org/Team%3AGroningen/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AGroningen/Demonstrate)
  + [Goal Vision](https://2019.igem.org/Team%3AGroningen/Vision)
* Parts
  + [Parts Overview](https://2019.igem.org/Team%3AGroningen/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AGroningen/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AGroningen/Composite_Part)
* [Safety](https://2019.igem.org/Team%3AGroningen/Safety)
* Human Practices
  + [Human Practices](https://2019.igem.org/Team%3AGroningen/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AGroningen/Public_Engagement)
  + [iGEM Search Engine](https://2019.igem.org/Team%3AGroningen/Search_Engine)
* Awards
  + [Software](https://2019.igem.org/Team%3AGroningen/Software)
  + [Hardware](https://2019.igem.org/Team%3AGroningen/Hardware)
  + [Measurement](https://2019.igem.org/Team%3AGroningen/Measurement)

# Notebook

1. [Cloning I](https://static.igem.org/mediawiki/2019/8/80/T--Groningen--Notebook3.pdf)
2. [Cloning II](https://static.igem.org/mediawiki/2019/b/b3/T--Groningen--Notebook12.pdf)
3. [Cloning III](https://static.igem.org/mediawiki/2019/d/df/T--Groningen--Notebook13.pdf)
4. [V. natriegens growth I](https://static.igem.org/mediawiki/2019/f/ff/T--Groningen--Notebook5.pdf)
5. [V. natriegens growth II](https://static.igem.org/mediawiki/2019/8/88/T--Groningen--Notebook11.pdf)
6. [Modelling](https://static.igem.org/mediawiki/2019/7/7a/T--Groningen--Notebook1.pdf)
7. [Fluorescence measurement](https://static.igem.org/mediawiki/2019/8/8d/T--Groningen--Notebook9.pdf)
8. [Vibrio transformation](https://static.igem.org/mediawiki/2019/d/df/T--Groningen--Notebook14.pdf)
9. [Stamping](https://static.igem.org/mediawiki/2019/2/22/T--Groningen--Notebook10.pdf)
10. [Bioink](https://static.igem.org/mediawiki/2019/5/53/T--Groningen--Notebook2.pdf)
11. [CRISPR](https://static.igem.org/mediawiki/2019/9/9f/T--Groningen--Notebook4.pdf)
12. [QR code formation, except bioprinting](https://static.igem.org/mediawiki/2019/f/fa/T--Groningen--Notebook6.pdf)
13. [Etching](https://static.igem.org/mediawiki/2019/8/87/T--Groningen--Notebook7.pdf)
14. [Encryption software](https://static.igem.org/mediawiki/2019/5/5b/T--Groningen--Notebook8.pdf)
15. [Laser Experiments](https://static.igem.org/mediawiki/2019/a/aa/T--Groningen--Notebook%22LASER_EXPERIMENTS%22.pdf)

# We thank our sponsors

[![DSM](https://static.igem.org/mediawiki/2018/4/40/T--Groningen--SponsorDSM.png "DSM")](https://www.dsm.com/corporate/home.html)
[![eurofins](https://static.igem.org/mediawiki/2018/thumb/d/d3/T--Groningen--eurofins2.png/1200px-T--Groningen--eurofins2.png "eurofins")](https://www.eurofins.nl/nl/)
[![BE-basic](https://static.igem.org/mediawiki/2018/b/b6/T--Groningen--SponsorBE-basic.png "BE-basic")](https://www.be-basic.org/)
[![education](https://static.igem.org/mediawiki/2019/8/8d/T--Groningen--SponsorScholierenAcademie.jpg "education")](https://www.rug.nl/education/scholierenacademie/)
[![Enteg](https://static.igem.org/mediawiki/2018/0/0c/T--Groningen--Sponsorenteg.png "Enteg")](https://www.rug.nl/research/enteg/)
[![GBB](https://static.igem.org/mediawiki/2018/7/70/T--Groningen--SponsorGBB.png "GBB")](https://www.rug.nl/research/gbb/)
[![GUF](https://static.igem.org/mediawiki/2018/a/a7/T--Groningen--SponsorGUF.png "GUF")](https://www.rug.nl/about-us/support-the-university/groninger-university-fund/)
[![jbi](https://static.igem.org/mediawiki/2018/3/3f/T--Groningen--Sponsorjbi.png "jbi")](http://www.cs.rug.nl/jbi/)
[![kivi](https://static.igem.org/mediawiki/2018/e/ee/T--Groningen--Sponsorkivi.png "kivi")](https://www.kivi.nl/)
[![qiagen](https://static.igem.org/mediawiki/2018/9/9b/T--Groningen--Sponsorqjagen.png "qjagen")](https://www.qiagen.com/nl/)
[![RIVM](https://static.igem.org/mediawiki/2018/a/a6/T--Groningen--SponsorRIVM.png "RIVM")](https://www.government.nl/ministries/ministry-of-health-welfare-and-sport)
[![SL](https://static.igem.org/mediawiki/2018/b/bb/T--Groningen--SponsorSL.png "SL")](https://www.rug.nl/sciencelinx/)
[![ziam](https://static.igem.org/mediawiki/2018/f/fa/T--Groningen--Sponsorziam.png "ziam")](https://www.rug.nl/research/zernike/)
[![Bioke](https://static.igem.org/mediawiki/2019/a/a5/T--Groningen--logos-bioke.png "Bioke")](https://www.bioke.com/)
[![De Jonge Onderzoekers](https://static.igem.org/mediawiki/2019/7/73/T--Groningen--SponsorDJOG.png "De Jonge Onderzoekers")](https://www.djog.nl/)
[![BD Kiestra](https://static.igem.org/mediawiki/2019/8/83/T--Groningen--SponsorBDkiestra.png "BD Kiestra")](https://www.icdrachten.nl/bedrijven/bd-kiestra)

##### iGEM Groningen 2019

Nijenborgh 7
9747AG Groningen
igem2019rug@gmail.com


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

# Team:Groningen/Experiments

iGEM Groningen 2019 :: Attributions

![iGEM Groningen](https://static.igem.org/mediawiki/2019/e/eb/T--Groningen--logo_transparent.png)

* [Home (current)](https://2019.igem.org/Team%3AGroningen)
* Team
  + [Team Members](https://2019.igem.org/Team%3AGroningen/Team)
  + [Collaborations](https://2019.igem.org/Team%3AGroningen/Collaborations)
  + [Sponsors](https://2019.igem.org/Team%3AGroningen/Sponsors)
  + [Attributions](https://2019.igem.org/Team%3AGroningen/Attributions)
* Project
  + [Description](https://2019.igem.org/Team%3AGroningen/Description)
  + [Design](https://2019.igem.org/Team%3AGroningen/Design)
  + [Experiments](https://2019.igem.org/Team%3AGroningen/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AGroningen/Notebook)
  + [Model](https://2019.igem.org/Team%3AGroningen/Model)
* Impact
  + [Results](https://2019.igem.org/Team%3AGroningen/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AGroningen/Demonstrate)
  + [Goal Vision](https://2019.igem.org/Team%3AGroningen/Vision)
* Parts
  + [Parts Overview](https://2019.igem.org/Team%3AGroningen/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AGroningen/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AGroningen/Composite_Part)
* [Safety](https://2019.igem.org/Team%3AGroningen/Safety)
* Human Practices
  + [Human Practices](https://2019.igem.org/Team%3AGroningen/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AGroningen/Public_Engagement)
  + [iGEM Search Engine](https://2019.igem.org/Team%3AGroningen/Search_Engine)
* Awards
  + [Software](https://2019.igem.org/Team%3AGroningen/Software)
  + [Hardware](https://2019.igem.org/Team%3AGroningen/Hardware)
  + [Measurement](https://2019.igem.org/Team%3AGroningen/Measurement)

# Experiments

### Recipes

##### Media

| Name | Components | Use |
| --- | --- | --- |
| LB | 15.5 g/L L broth (Formedium) | Growth of *E. coli* |
| LBV / LB3 | LB medium supplemented with 30 g/L NaCl | Growth of *V. natriegens* |
| BHI v2 | 37 g/L BHI broth (Difco) v2 salts | Growth of *V. natriegens* |
| BHI v3 | 37 g/L BHI broth (Difco) v3 salts | Growth of *V. natriegens* |
| LB v2 | LB medium supplemented with v2 salts | Growth of *V. natriegens* |
| LB v3 | LB medium supplemented with v3 salts | Growth of *V. natriegens* |
| *V. natriegens* minimal medium | 2 g/L Na D-glucuronate  1.3 g/L MgSO4·7 H2O  0.75 g/L KCl  5.3 g/L (NH4)2HPO4  15 g/L NaCl | Growth of *V. natriegens* |
| M9 | 11.28 g/L 5 x M9 salt base (Formedium)  0.4 % glucose  2 mM MgSO4  0.1 mM Cacl2  0.3 µM thiamine·HCl | Growth of *E. coli* in CRISPR screen |
| M9 his | M9 medium supplemented with 3 mM L-histidine | Growth of *E. coli* in CRISPR screen |
| M9v2 | M9 medium supplemented with v2 salts | Growth of *V. natriegens* in CRISPR screen |
| M9v2 his | M9v2 medium supplemented with 3 mM L-histidine | Growth of *V. natriegens* in CRISPR screen |
| *V. natriegens* recovery medium | BHIv2 supplemented with 680 mM sucrose | Recovery of *V. natriegens* after transformation |
| Printing medium | 0.5 mM Cacl2 | Plates for printing with bioink |

### Media supplements

| Name | Amount | Use |
| --- | --- | --- |
| Agar | 10 g/L | Agar plates |
| v2 salts | L204 mM NaCl  4.2 mM KCl  23.14 mM MgCl2 | Growth of *V. natriegens* |
| v3 salts | 475 mM NaCl  9.7 mM KCl  23.14 mM MgCl2 | Growth of *V. natriegens* |
| Kanamycin | *E. coli*: 50 µg/ml  *V. natriegens*: 250 µg/ml | Antibiotic selection |
| Chloramphenicol | *E. coli*: 25 µg/ml  *V. natriegens*: 25 µg/ml | Antibiotic selection |
| Ampicillin | *E. coli*: 100 µg/ml  *V. natriegens*: 70 µg/ml | Antibiotic selection |
| Tetracycline | *E. coli*: 10 µg/ml  *V. natriegens*: 4 µg/ml | Antibiotic selection |
| Anhydrotetracycline | 500, 250, 125 or 62.5 ng | Induction of promoter |
| L-Arabinose | 1, 0.5, 0.1, 0.05 or 0.01 (w/v) % | Induction of promoter |
| Mannose | 1, 0.5, 0.1 or 0.05 (w/v) % | Induction of promoter |

### Other recipes

| Name | Components / Instructions | Use |
| --- | --- | --- |
| 10 x TAE | 48.4 g/L Tris base  17.4 M concentrated acetic acid  3.7 g/L EDTA | Buffer for gel electrophoresis |
| Agarose gel | 1 % Agarose dissolved in TAE by heating  10000 x diluted DNA stain stock | Gel for gel electrophoresis |
| *V. natriegens* electroporation buffer | 680 mM sucrose  7 mM KPi | Preparation of electrocompetent *V. natriegens* |
| Sorbitol | 1 M sorbitol | Preparation of chemically competent *V. natriegens* |
| Calcium chloride | 0.1 M Cacl2 | Preparation of chemically competent *E. coli* |
| Calcium chloride, glycerol | 0.1 M Cacl2  15 % glycerol | Preparation of chemically competent *E. coli* |
| Sodium Citrate | 0.1 M Na Citrate | Resolubilization of solidified bio-ink |

### Procedures

##### 1) Molecular genetic methods

Gel electrophoresis

1. Make an agarose gel using an adequate mold and comb
2. Load 10 µl of the sample in the pocket alongside the DNA ladder
3. Apply 120 V for 30 min
4. Image the gel using gel documentation machine

Digestion ligation cloning

1. Design the cloning procedure using the Benchling Assembly tool
2. Digest the desired parts with the respective enzymes according to the fabricant’s instructions
3. Inactivate the restriction enzymes
4. Ligate all parts according to the fabricant’s instructions
5. Inactivate the T4 ligase
6. Transform 5 µl of the ligated construct according to protocol
7. Select clones and confirm by cPCR and sequencing

Preparation of electrocompetent *V. natriegens* cells

1. Dilute an overnight culture 1:200 in LBv2 and culture to an OD600 of 0.4 – 0.6 (use 1.5 ml culture per aliquot)
2. Pellet cells 3 min at 8000 rpm
3. Resuspend the cells in 1 ml of electroporation buffer
4. Repeat steps 2 and 3
5. Pellet cells and resuspend in electroporation buffer (30 µl per aliquot)

Electroporation of *V. natriegens* cells

1. Add 300 ng of DNA to electrocompetent cells and transfer into 1 mm electroporation
2. Electroporate at 900 V, 25 µF, 200 Ω
3. Recover with 1 ml recovery medium and culture for 1.5 h at 37 °C
4. Plate on LBv2 plates with according antibiotic marker

Preparation of chemically competent *V. natriegens* cells

1. Grow overnight at 30 °C, 275 rpm in BHIv2
2. Dilute the overnight culture 1:200 in BHIv2 (100 ml) and grow at 37 °C in a baffled flask to an OD600 of 0.4
3. Pellet the cells at 6465 rpm for 10 min
4. Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
5. Use 200 µl per aliquot
6. Use the same day or freeze in -80 °C

Preparation of chemically competent *V. natriegens* cells

1. Grow overnight at 30 °C, 275 rpm in BHIv2
2. Dilute the overnight culture 1:200 in BHIv2 (100 ml) and grow at 37 °C in a baffled flask to an OD600 of 0.4
3. Pellet the cells at 6465 rpm for 10 min
4. Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
5. Use 200 µl per aliquot
6. Use the same day or freeze in -80 °C

Chemical transformation (*V. natriegens*)

1. Thaw chemically competent cells on ice
2. Add approx. 50 ng of DNA (not more than 10 % of the cell Volume) and flick to combine
3. Incubate 10 min on ice
4. Transfer to 42 °C water bath for 2.5 min
5. Recover with 2.5 ml prewarmed recovery medium and let outgrow for 2 h in 37 °C
6. Plate on LBv2 plate with respective antibiotic

Colony PCR (cPCR)

1. Pick a colony using a toothpick
2. Use the toothpick to first squeeze the colony at a bottom of a PCR tube and then inoculate liquid culture
3. Add the PCR reaction mix and start the PCR reaction according to the fabricant’s instructions
4. Analyze the PCR products using gel electrophoresis

CRISPR

1. *Target selection and design*
   The gene to knock out was selected based on literature on auxotrophic *E. coli* strains. gRNA binding sites were designed using the Synthego software. We selected one gene to knockout and three gRNA targets. For homologous recombination a 1.5 kb long part up- and downstream of the HisD gene were selected and brought together.
2. *CRISPR plasmid cloning*
   The pJOE8999 plasmid was digested using BsaI restriction enzyme and verified on an agarose gel. pJOE8999 contains the Cas9 enzyme under the control of the mannose inducible promoter, gRNA scaffold with two BsaI restriction sites and kanamycin resistance marker. The gRNAs were ordered as single strand nucleotides from IDT and annealed by cooling 0.1 °C/s from 95 °C to 25 °C. The annealed insert was designed in such a way that it would match the sticky ends created by the BsaI enzyme. The gRNA insert was ligated into the plasmid using the ligation protocol. The correct construct was confirmed by sequencing.
3. *Homologous part*
   The homologous part of 3 kbp was ordered as gBLOCKS from IDT. This double stranded DNA fragment was shortened to 1.5 kbp and 600 bp respectively using PCR and according primers. The PCR was conducted using Q5 enzyme and following the fabricant’s instructions. Successful PCR was validated with gel electrophoresis and the fragments were isolated using Qiagen’s PCR purification kit.
4. *CRISPR knockout*
   The constructed plasmid and homologous part were transformed into *E. coli* and *V. natriegens* using the respective protocols and plated on either LB or LBv2 agar plates supplemented with kanamycin and the inducing agent mannose (0.1 %) to encourage Cas9 expression. Clones that have appeared within 48 h in 30 °C were plated on LB or LBv2 agar plates without the selection marker kanamycin to eliminate the plasmid. Since this plasmid contains a heat sensitive origin of replication usually the bacteria would be incubated in 50 °C. However, we decided against this because of *V. natriegens*’ sensitivity to heat.
5. *Screening*
   Colonies growing on non-selective agar plates were replated on M9 agar plates either with or without histidine. If a successful CRISPR mediated HisD knockout leads to a histidine auxotrophic strain the modification can be identified if the clone was able to grow only on the plate that had been supplemented with histidine.

Synthetic promoter library (SPL) construction

1. *Target selection and design*
   The promoters pTET (BBa\_R0040) and pBAD (BBa\_K808000) were selected for optimization because they have been reported to be less inducible in *V. natriegens* than reported in E.coli. The SPL was designed following the work of 2013 iGEM DTU-Denmark team.
2. *SPL cloning*
   Both promoters were constructed to control the expression of mCherry. The SPL was achieved by PCR of these “native” constucts with randomized primers that yield an arbitrary sequence in the promoter region. The linear PCR product was circulized with the digestion ligation method. The random plasmids were transformed in both *E. coli* and *V. natriegens*.
3. *Screening*
   Clones were picked and recultured. In an initial screening all clones were measured for fluorescence of mCherry after induction with anhydro tetracycline or arabinose. Clones with at least five times inducibility were selected and remeasured for mCherry fluorescence in duplicate. Selected clones were characterized in different inducer concentrations in a time course measurement.

##### 2) Production of QR code pattern

Etching

1. Seed a lawn of bacteria on the plate: leave 15 ml of culture with OD600 of 0.2 for 15 min. Then remove excess liquid and let dry for 10 more minutes
2. Using a CNC laser plotter machine (400 - 460 nm, 500 mW) at a speed of 250 mm/min or 550 mm/min and density of 12 or 14 lines per mm, etch the desired image into the bacterial lawn (variables will vary depending on organism used, these values have worked for us with *E. coli* and *V. natriegens* respectively)
3. Let the plate outgrow at 37 °C for 24 h and admire the beautiful image in etched into the bacterial lawn

Stamping

1. 3D print a stamp (use QRoningen software)
2. Dilute fresh culture to an OD600 of 0.15 and dip the stamp in it
3. Let excess liquid flow back and let stamp dry for 15 min
4. Touch the stamp to an agar plate (you can stamp around five plates after one another)
5. Do not move the plate and let it dry for 10 more minutes
6. Incubate the plate overnight at 37 °C and enjoy a gorgeous bacteria stamp

Bioink

1. Pellet an overnight culture at 4000 rpm for 3.5 min
2. Resuspend carefully in LB or LBV supplemented with alginate.

Viability in bioink

1. Print a drop of 100 µl from the bio-ink in the center of a printing medium plate
2. Leave the plate for 6, 24 and 72 h on the benchtop
3. Resolubilize the drop after the given incubation time in 0.1 M sodium citrate solution, make a serial dilution and use 10 µl each to plate on a LB or LBV plate
4. Let grow overnight and count the appeared colonies to determine CFU

Bioprinting

1. Prepare fresh bio-ink and printing plates (supplemented with Cacl2) and the bioprinter
2. Create the gcode for your QR code using QRoningen software
3. Start the bioprinter to run your gcode
4. Incubate plates at 37 °C

##### 3) Measurements

Fluorescence in liquid medium

1. Grow overnight cultures of the bacteria you want to measure
2. Inoculate in enough fresh growth medium in the morning. If needed supplement with inducers at different concentrations.
3. Transfer 200 µl / well to 96-well plate. Remember to make a duplicate measurement. Measure OD600 and the fluorescence of your reporter in a microtiter plate reader.

   Type | Absorption / Emission || mCherry | ex.: 580 nm, em.: 630 nm |
   | RFP | ex.: 520 nm, em.: 580 nm |
   | GFP | ex.: 485 nm, em.: 528 nm |
4. Let cultures grow in 37 °C
5. Transfer to 96-well plate at desired timepoints to quantify the development over time (e.g. 4 and 8 h) and measure

Fluorescence in solid culture

1. Produce a fresh plate with cultures, either in bio-ink or regular agar plates
2. Measure fluorescence of your reporter using the Typhoon FLA 9500 biomolecular imager.

   Type | Excitation wavelength || mCherry | Alexa Fluor 532 nm excitation wavelength |
   | RFP | Cy3 532 nm excitation wavelength |
   | GFP | Alexa Fluor 478 nm excitation wavelength |

Data processing (solid)

1. Process the image using ImageJ
2. Select area of choice that contains the fluorescent reporter
3. Determine average intensity in samples and controls

Data processing (liquid)

1. Process data in any data processing software (e.g. Excel)
2. Normalize fluorescence with OD600 (Fnorm = F / OD600)
3. Determine foldchange to your control (FC = Fnorm / Fnorm,control)
4. Plot fluorescence against time and inducer concentration if applicable

# We thank our sponsors

[![DSM](https://static.igem.org/mediawiki/2018/4/40/T--Groningen--SponsorDSM.png "DSM")](https://www.dsm.com/corporate/home.html)
[![eurofins](https://static.igem.org/mediawiki/2018/thumb/d/d3/T--Groningen--eurofins2.png/1200px-T--Groningen--eurofins2.png "eurofins")](https://www.eurofins.nl/nl/)
[![BE-basic](https://static.igem.org/mediawiki/2018/b/b6/T--Groningen--SponsorBE-basic.png "BE-basic")](https://www.be-basic.org/)
[![education](https://static.igem.org/mediawiki/2019/8/8d/T--Groningen--SponsorScholierenAcademie.jpg "education")](https://www.rug.nl/education/scholierenacademie/)
[![Enteg](https://static.igem.org/mediawiki/2018/0/0c/T--Groningen--Sponsorenteg.png "Enteg")](https://www.rug.nl/research/enteg/)
[![GBB](https://static.igem.org/mediawiki/2018/7/70/T--Groningen--SponsorGBB.png "GBB")](https://www.rug.nl/research/gbb/)
[![GUF](https://static.igem.org/mediawiki/2018/a/a7/T--Groningen--SponsorGUF.png "GUF")](https://www.rug.nl/about-us/support-the-university/groninger-university-fund/)
[![jbi](https://static.igem.org/mediawiki/2018/3/3f/T--Groningen--Sponsorjbi.png "jbi")](http://www.cs.rug.nl/jbi/)
[![kivi](https://static.igem.org/mediawiki/2018/e/ee/T--Groningen--Sponsorkivi.png "kivi")](https://www.kivi.nl/)
[![qiagen](https://static.igem.org/mediawiki/2018/9/9b/T--Groningen--Sponsorqjagen.png "qjagen")](https://www.qiagen.com/nl/)
[![RIVM](https://static.igem.org/mediawiki/2018/a/a6/T--Groningen--SponsorRIVM.png "RIVM")](https://www.government.nl/ministries/ministry-of-health-welfare-and-sport)
[![SL](https://static.igem.org/mediawiki/2018/b/bb/T--Groningen--SponsorSL.png "SL")](https://www.rug.nl/sciencelinx/)
[![ziam](https://static.igem.org/mediawiki/2018/f/fa/T--Groningen--Sponsorziam.png "ziam")](https://www.rug.nl/research/zernike/)
[![Bioke](https://static.igem.org/mediawiki/2019/a/a5/T--Groningen--logos-bioke.png "Bioke")](https://www.bioke.com/)
[![De Jonge Onderzoekers](https://static.igem.org/mediawiki/2019/7/73/T--Groningen--SponsorDJOG.png "De Jonge Onderzoekers")](https://www.djog.nl/)
[![BD Kiestra](https://static.igem.org/mediawiki/2019/8/83/T--Groningen--SponsorBDkiestra.png "BD Kiestra")](https://www.icdrachten.nl/bedrijven/bd-kiestra)

##### iGEM Groningen 2019

Nijenborgh 7
9747AG Groningen
igem2019rug@gmail.com

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

# Team:Groningen/Results

iGEM Groningen 2019 :: Results

![iGEM Groningen](https://static.igem.org/mediawiki/2019/e/eb/T--Groningen--logo_transparent.png)

* [Home (current)](https://2019.igem.org/Team%3AGroningen)
* Team
  + [Team
    Members](https://2019.igem.org/Team%3AGroningen/Team)
  + [Collaborations](https://2019.igem.org/Team%3AGroningen/Collaborations)
  + [Sponsors](https://2019.igem.org/Team%3AGroningen/Sponsors)
  + [Attributions](https://2019.igem.org/Team%3AGroningen/Attributions)
* Project
  + [Description](https://2019.igem.org/Team%3AGroningen/Description)
  + [Design](https://2019.igem.org/Team%3AGroningen/Design)
  + [Experiments](https://2019.igem.org/Team%3AGroningen/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AGroningen/Notebook)
  + [Model](https://2019.igem.org/Team%3AGroningen/Model)
* Impact
  + [Results](https://2019.igem.org/Team%3AGroningen/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AGroningen/Demonstrate)
  + [Goal
    Vision](https://2019.igem.org/Team%3AGroningen/Vision)
* Parts
  + [Parts
    Overview](https://2019.igem.org/Team%3AGroningen/Parts)
  + [Basic
    Parts](https://2019.igem.org/Team%3AGroningen/Basic_Part)
  + [Composite
    Parts](https://2019.igem.org/Team%3AGroningen/Composite_Part)
* [Safety](https://2019.igem.org/Team%3AGroningen/Safety)
* Human Practices
  + [Human
    Practices](https://2019.igem.org/Team%3AGroningen/Human_Practices)
  + [Education &
    Engagement](https://2019.igem.org/Team%3AGroningen/Public_Engagement)
  + [iGEM
    Search
    Engine](https://2019.igem.org/Team%3AGroningen/Search_Engine)
* Awards
  + [Software](https://2019.igem.org/Team%3AGroningen/Software)
  + [Hardware](https://2019.igem.org/Team%3AGroningen/Hardware)
  + [Measurement](https://2019.igem.org/Team%3AGroningen/Measurement)

# Results

At the start of the project we set out several goals so our eventual protocol would be easy to use,
robust, reproducible and safe.

**Goals:**

* The protocol and software accompanying the protocol should be easy to use.
* The developed method should give consistent results.
* Several strains have to be used in the same code for the code to be hard to crack.
* During shipment, the code should not be visible, and only upon incubation under the right
  circumstances the code needs to becomes visible.

**Achievements:**

* We created a protocol for precise bacterial disposition using 3D-printed stamps and a
  modified 3-D printer.
* We have developed custom alginate based bio-ink that forms a hydrogel upon contact with
  a
  calcium coated surface.
* By printing on a base without nutrients, we prevent the bacteria from leaving the
  hydrogel
  increasing the precision of our protocol.
* We show that *Escherichia coli* and *Vibrio natriegens* stay viable inside the
  printed hydrogel when printed on an agar base without additional nutrients in several
  conditions.
* We show that modified *E. coli* in the hydrogel are inducible with chemicals when
  stored at 4°C for several days.
* Our software[link to software] is easy to use and by providing the program with the
  information you want to encrypt and a password, an AES encrypted QR-code will be
  generated
  in computer aided design (CAD) software.
* This QR-code can then be converted to G-code which can be used by our modified
  3D-printer.
* Our in-house developed pattern recognition software is then able to scan the QR code
  using
  any camera. Upon supplementation of the password, the message will be revealed.

## 1. Bacterial Deposition

For consistency, we need to be able to deposit genetically engineered bacteria in the shape of a
QR
code with high precision. For this to become reality we explored several methods of pattern
creation
namely etching, stamping and printing.

### 1.1 Etching

After trying out several protocols and settings, we were eventually able to use a laser engraver
to etch a QR code into a plate with bacteria. Here the bacteria are spread over the surface of
the plate before being treated with a laser. After incubation overnight, the pattern appears
(Figure 1). This method produces readable QR codes.

![Etched Plate](https://static.igem.org/mediawiki/2019/thumb/4/47/T--Groningen--Result1.jpg/1200px-T--Groningen--Result1.jpg)

Figure 1 - Etched QR codes in a plate with *E.coli*, the pink colour comes from
J0445.

However, anyone that gets these plates would be able to scan the code. For our QR code to be
secure, several strains need to be used that become visible or grow under certain conditions.
This is not employable using this method. In any case, we show that a laser can be used to kill
bacteria with high precision.

### 1.2. Stamping

To further test methods for precise bacterial deposition, stamps were explored. The stamps were
rested in a solution with bacteria before tapping it gently on an agar plate. The first
generation of 3D printed stamps were solid (Figure 2).

![Etched Plate](https://static.igem.org/mediawiki/2019/thumb/9/96/T--Groningen--Result2.png/1200px-T--Groningen--Result2.png)

Figure 2 - the four generations of QR codes from left to right: first, second,
third, fourth

Ultimately this resulted in blurry codes (Figure 3). The next generations of 3D printed stamps
therefore were pixelated, allowing excess fluid to flow away in the grooves eventually resulting
in a clearer, readable QR code (figure 4).

![Etched Plate](https://static.igem.org/mediawiki/2019/3/33/T--Groningen--Result3.png)

Figure 3 - On the left are the results of stamps without grooves and on the
right side are the pixelated stamps.

Additionally, we tried out prongs to be able to correctly orientate several stamps, using
several strains of bacteria (figure 2). This is needed to increase the security of our
procedure. However, we ran into problems with manually aligning the stamps regardless of the
prongs. Printing one stamp takes about 3 hours. This makes the whole procedure quite
time-consuming and far from easy to use, especially in the case of multiple stamps per code.
These issues prompted us to further develop other methods in our pipeline, but not before we
were able to scan a code using our image processing software [link to software] (figure 4).

![Black and White QR](https://static.igem.org/mediawiki/2019/c/c6/T--Groningen--Result4.jpg)

Figure 4 – Stamped plate, processed with our software.

### 1.3. Printing of bio-ink using a 3D printer

#### 1.3.1. Conversion of a 3D-printer to a bioprinter

In order to print a biological QR code, we set out to modify a commercially available 3D
printer (Vertex K8400, into a bioprinter (link to hardware). First the filament extruder
head, the part that pushes the filament to nozzle, is replaced with a peristaltic pump head
(figure 4). A pipette tip (10 µL tip) connected to silicon tubing (inner diameter of 1.6 mm)
was mounted on top of the print head (figure 5). For more than one type of bio-ink, we can
potentially use the second extruder of the printer and modify it similarly. The silicon
tubing is plugged in the reservoir via the peristaltic pump head, setting the printer up for
handling liquid biological ink (bio-ink). This bio-ink solidifies within seconds upon
contact with the printing surface (link to bio-ink on this page). The instructions (G-code)
responsible for instructing the printer to create a QR code can be created using our custom
software (link to software). We can control the stepper motor driving the peristaltic pump
head to control and adjust the flow rate of bio-ink during printing jobs. The temperature
requirements for printing are removed from the G-code to allow the printer to print without
heating the nozzle. These adaptations effectively set up our printer for printing multiple
engineered bacteria using bio-ink.

![Black and White QR](https://static.igem.org/mediawiki/2019/a/a8/T--Groningen--Result5.png)

![Black and White QR](https://static.igem.org/mediawiki/2019/2/2b/T--Groningen--Result6.png)

Figure 5 - upper left, filament extruder head, upper right, peristaltic pump head, bottom left, pipette tip attached to print head, bottom right, silicone tubing running from print head to reservoir

#### 1.3.2. Resolution and reproducibility of the printer.

Our printer needs to be able to consistently print squares, since they are in every QR
code. The consistency of the printer was determined by printing a square inside a square
in a petri-dish using the developed bio-ink [link to bio-ink]. Food dye was added to the
ink to ensure visibility. Without changing any parameters in the G-code, we printed four
plates consecutively as shown in figure 6. What can be seen is that at the beginning of
a line, more ink is deposited on the plate. This effect is increased due to the
beginning and end of these lines coinciding, creating a blob (figure 6). Provided these
discrepancies are not to big, we believe that this would not prevent a QR code from
being readable. We demonstrate that our printer can print squares with proper
consistency needed for our projects aim.

![Black and White QR](https://static.igem.org/mediawiki/2019/5/54/T--Groningen--Result7.jpg)

Figure 6 - Reproducibility of bioprinter

To characterize the spatial resolution of printed structures, 2 circles and a square
were printed to evaluate the divergence in the same print and evaluate the resolution.
The line width was measured using ImageJ Fiji software package (imageJ) and is shown in
figure 7. The line width generated by our printer varies. At the beginning the line is
thicker due to the blob. Ultimately we were able to obtain line widths as low as 0.5 mm.
Optimization of the extrusion parameter is potentially a way to decrease the variation
in the line width. However, having a consistent line width is not necessarily required
for the end goal of our project.

![Black and White QR](https://static.igem.org/mediawiki/2019/a/aa/T--Groningen--Result8.png)

Figure 7 - Line width measurements

Because we have control of the flow, we are able to print dots. The liquid droplets can
spread. When dots are printed to close to each other they can merge (figure 7). We found
that printing within 2 mm of each other can sometimes allow them to merge, since
alginate cross-linking is not instantaneous. Potentially this can be prevented when
increasing the time between dots that are close to each other. We used our software
(link to software) to convert the word “QRoningen’’ into a QR-code and transform the
code to G-code instructions for the printer. After several tries, and modifying the
extrusion parameters in the G-code, we were able to print a QR-code.

![Black and White QR](https://static.igem.org/mediawiki/2019/9/92/T--Groningen--Result9.jpg)

Figure 8 - QR code printed using food dye stained bio-ink on calcium
chloride agar

#### 1.3.3. Bio-ink

Bio-ink was developed to allow for printing using mild conditions suitable for live
bacteria. Sodium alginate inside the ink supplemented with nutrients is in liquid
form when moving through the tubing but solidifies upon contact with calcium ions in
the printing surface. The calcium ions cross-link the alginate molecules forming a
stable gel within seconds [2]. Several concentrations of alginate (1 % w/v to 10 %
w/v) in the ink and CaCl2 (0.05 – 5 M) in the printing surface were varied to find
optimal values [1]. We also found that low concentrations of alginate would result
in poor gel quality (below 1.5 %) and high concentrations of alginate resulted in
viscous ink that would limit control of the flow or was impossible to move. Very
high concentrations of CaCl2 prevented agar from becoming solid (over 1M) and would
increase the speed of solidification in addition to being undesirable for bacteria
due to osmotic stress. Furthermore, these high concentrations would allow for the
ink to solidify in the tip, preventing extrusion and decreasing the robustness of
the procedure. At concentrations as low as 0.05 M CaCl2 we found that the speed of
solidification was still within seconds when using small volumes. Hence, we use 0.05
M CaCl2 in the printing surface and 2 % w/v sodium alginate in the ink in our
printing procedures.

#### 1.3.4. Viability of *E. coli* and *V. natriegens* in printed bio-ink.

The bacteria inside need to be able to survive when incorporated in the printed
ink. To evaluate this, *E. coli* and *V. natriegens* were printed on
agar with
nutrients using our custom ink to evaluate the survivability. After storing
unprinted ink and plates with the printed bacteria from 6 to 72 hours at room
temperature, samples were treated with a sodium citrate solution to resolubilize
the gels. The amount of bacteria inside the samples were evaluated using the
plate count method (Figure 9). Interestingly, we found that *E. coli*
exhibits
almost no growth when inside the unprinted gels. Moreover, *V natriegens*
seems to
become less viable over time when stored inside bacterial ink. The hindered
growth of both organisms could be explained by the absence of shaking of the
tubes, reducing aeration. These findings suggest it is best to use freshly
prepared ink for printing.
When printed on a supporting structure that has extra nutrients, bacterial
growth is observed in both organisms (Figure 9). A ring of bacteria was observed
around the gels. Bacteria were also observed on top of the printed gels. The
nutrients inside the agar potentially allow for the bacteria to grow and move
outside of the bounds of the gel and grow further. This overgrowth of bacteria
is unwanted in the case of QR-code printing, since it could disturb the
readability of the code.

![Black and White QR](https://static.igem.org/mediawiki/2019/4/48/T--Groningen--Result10.png)

Figure 9 – Viability assay of *E. coli* and *V.
natriegens* on
plates with nutrients, stored at room temperature.

By supplying no additional nutrients in the plate we aimed at confining the
bacteria to their initial printed position. The plates with the printed ink were
kept for varying amounts of time ranging from 0 h to up to one week at room
temperature before a viability assay was performed (Figure 10). In the first 24
hours growth is observed in both organisms. Thereafter no growth was observed.
These findings are in line with previous work [1], and suggest that the
organisms keep growing until a cap is achieved, after which the limited amount
of space and nutrients, provided in the ink limits growth. Indeed, here we show
that the printed gels are capable of supporting growth of bacteria, without
additional nutrients.

![Black and White QR](https://static.igem.org/mediawiki/2019/d/d6/T--Groningen--Result11.png)

Figure 10 – Viability assay *E. coli* and *V.
natriegens* in ink on
plates without nutrients stored at room temperature.

For the purpose of our project it is beneficial that the code is not visible
until induced. We anticipated that by storing the plates with printed ink in the
fridge at 4°C for 3 days, we could halt bacterial growth until growth is needed.
Another viability assay was performed to confirm this (figure 11). Note that
there are no nutrients inside the plate and that the vibrio culture was grown
for only 6 hours prior to be made into ink. The viability of *E. coli* dropped 3
fold and the viability of *V. natriegens* dropped 4 fold when stored at 4°C for 3
days inside printed ink. This fold change is bigger than is observed in liquid
culture in literature when *V. natriegens* is kept at 4°C [3]. Potentially this is
caused by the limited amount of nutrients. Here we show that the bacteria stay
viable when stored at at 4°C, for example during transport. This can be used to
prevent the code from appearing during transport.

![Black and White QR](https://static.igem.org/mediawiki/2019/5/5c/T--Groningen--Result12.png)

Figure 11 – Viability assay of *E. coli* and *V.
natriegens* inside printed gels stored at 4°C on plates without
nutrients.

#### 1.3.5. Inducibility inside the printed ink.

**1.3.5.1. Bio-film induction**

Qroningen hinges on inducibility of constructs inside genetically engineered bacteria inside
our printed alginate gels. Dr Srikkanth Balasubramanian (link to attributions) was kind
enough to send us two plasmids used to show ability of biofilm formation inside the physical
support of printed alginate hydrogels [4]. One plasmid encoding for pSB1C3-GFP-pRha-CsgA
allowing for rhamnose inducible CsgA production accompanied by constitutive GFP production.
The other plasmid encoded for pSB1C3-GFP offering only constitutive GFP production.
Bacterial ink harboring these plasmids were deposited on print plates with rhamnose (0.5 %).
The experiment was performed on plates with inducer and plates without inducer. After
induction, the plates were treated by adding a 0.1 M solution of sodium citrate and
incubating at room temperature for 3 hours at 70 rpm. The liquid was removed after 3 hours
effectively removing the printed gels from the plates. After treatment, the plates were
observed using a camera and the Typhoon FLA 9500 biomolecular imager (figure 12 and 13).
Biofilm formation can be observed on plates that have the plasmid and the inducer after 2
days. Biofilm manifests itself as a thin film where the alginate gel used to be. On plates
without additional nutrients no biofilm formation is observed after 6 days

![Black and White QR](https://static.igem.org/mediawiki/2019/thumb/4/45/T--Groningen--Result13.png/1200px-T--Groningen--Result13.png)

Figure 12 – Imaged plates after 3 days, before and after treatment with 0.1M
sodium citrate.

![Black and White QR](https://static.igem.org/mediawiki/2019/thumb/a/af/T--Groningen--Result14.png/1200px-T--Groningen--Result14.png)

Figure 13 – Image using Typhoon FLA 9500 biomolecular image using Alexa
Fluor 488 settings.

The induced CsgA protein subunits are transported outside of the bacteria where they
self-assemble into CsgB proteins that form curli amyloid fibers [5]. These form the protein
core of the extracellular biofilm native to *E. coli* [4–6]. These findings suggest that a
considerable amount of nutrients is needed for formation of such a biofilm. More
importantly, these findings show that substances inside the alginate gel can diffuse from
the printing substrate into the alginate gel and exert an effect on the resident bacteria.

**1.3.5.2. GFP and RFP fluorescence when inducers are supplemented to the plate at a later time point.**

In the previous experiment, the inducer was in the supporting structure. For our
application it would be ideal if inducers could be added later. This was evaluated by
depositing *E. coli* harboring a tetracycline inducible green fluorescent protein GFP on
print plates with nutrients [link to sonja, attributions]. A certain amount of ink was
deposited on the plate. Later, an equivalent of liquid containing commonly used
concentrations of anhydrous tetracycline (aTC) and tetracycline (TC) were added to the
plate. After 24 hours of incubation, the plates were observed in the typhoon FLA 9500
biomolecular imager and the fluorescence was quantified using imageJ. The area of
interest was selected and measured in triplicate for each continuous area of cells on
each sample. The results of which are shown in figure 14. This indicates that inducers
can be added later to the plate and are able to diffuse through the printed ink.

![Black and White QR](https://static.igem.org/mediawiki/2019/5/5a/T--Groningen--Result15.png)

Figure 14 – Quantification of fluorescence in printed ink imageJ. Plates
were induced immediately, incubated for 24 hours and observed.

For our application, it would be ideal to send a plate in refrigerated conditions, add
an inducer and then observe the plate. In this experiment, *E. coli* containing a
tetracycline inducible red fluorescent protein (RFP) were printed using a pipette on a
printing plate containing no additional nutrition in triplicate [link to sonja,
attributions]. A less-toxic tetracycline derivative aTC, was added 48 hours after
storage at 4°C as inducer. The plates were incubated and imaged 24 hours later using the
typhoon FLA 9500 biomolecular imager. The fluorescence was quantified with imageJ.
Fluorescence intensity was measured by measuring the average intensity of a fixed area
with bio-ink in triplicate (Figure 15). What can be seen is that the intensity of the
plates that have the construct are higher, this is probably due to leakiness of the
promoter. Furthermore, when 1 µg/mL was used, a significant increase in fluorescence can
be observed thus indicating our proposed method is viable. Note that the amount of
inducer used here to elicit a response was higher than in the previous experiment. This
can be explained because bacteria become less responsive over time. We would have to
carefully evaluate inducer concentrations of all of the constructs we use. Non-toxic
inducers are preferred for this reason.

![Black and White QR](https://static.igem.org/mediawiki/2019/4/4a/T--Groningen--Result16.png)

Figure 15 – Quantified fluorescence measurement using imageJ inside
bioink that was stored for 2 days at 4°C. The ink was induced with aTC,
incubated overnight and imaged 24 hours later. At 1 µg /mL fluorescence is
observed.

Overall these experiments show that our concept is achievable. We demonstrate that we
can deposit bacteria using a modified 3D-printer as dots instead and squares. This gives
us spatial control over bacterial deposition. A feat that, to the best of our knowledge,
has not yet been achieved before using a modified commercial 3D printer. We have also
shown that bacteria are able to survive inside the ink in refrigerated conditions,
staying responsive to inducers. Inducers are able to diffuse through the printed gel and
can therefore be added later to an already printed plate. Not using nutrients in the ink
allows us to prevent bacteria from leaving their initial place on the plate preserving
the resolution of the code over time. In multiple experiments using diverging constructs
we show that genetically engineered *E. coli* cells are inducible inside our printed
bio-ink.

## 2. Genetic engineering and characterization

### 2.1. Characterization of promoters and constructs

The characterization of our constructs focuses on inducible promoters. These regulatory parts are of utmost importance to our project as they allow for environmentally controlled phenotypes. Only if the correct key is added - in this case inducers - the QR code will be readable.

#### 2.1.1. Constructs in *V. natriegens*

Because of its astonishing growth rate of only 7 minutes, *V. natriegens* is ideal for our purposes. The quicker growth means a quicker readout of the message. A lot of procedures are not optimized yet for use in *V. natriegens* though, so we decided to use our project to also help with the popularization of this intriguing organism and provide a step forward in exploring all its possibilities. We characterized the inducible promoters using mCherry as reporter as we were advised by *V. natriegens* expert Tanya Tschirhart.

##### 2.1.1.1. P1 Promoter

We contributed a new basic part, the native P1 promoter from *V. natriegens* (BBa\_K3171171). The gene under the control of this promoter is transcribed constitutively. Fluorescence measurements of the construct in *E. coli* and *V. natriegens* reveal a constitutive expression of the reporter mCherry for both organisms. Interestingly our measurements show that the expression levels in *E. coli* are a lot higher than in *V. natriegens* as the fold change is 225 for *E. coli* compared to 3 for *V. natriegens*.

![Black and White QR](https://static.igem.org/mediawiki/2019/3/37/T--Groningen--Result17.png)

Figure 16 – Fluorescence of the reporter mCherry under the control of the constitutive P1 promoter in *V. natriegens* and *E. coli* in comparison to control without plasmid.

##### 2.1.1.2. pBAD in *V. natriegens*

The pBAD promoter in part BBa\_K808000 is one of the most commonly used parts in iGEM and synthetic biology. We characterized its use and inducibility in *V. natriegens*. Our measurements show very high expression levels for a concentration of 0.5 % (w/v) arabinose and a very low basal level of expression without the inducer and the fold change amounts to 1127. In conclusion, pBAD offers a tight and regulation of expression in *V. natriegens*.

![Black and White QR](https://static.igem.org/mediawiki/2019/0/0d/T--Groningen--Result18.png)

Figure 17 – pBAD is a tightly regulated promoter that can be induced by arabinose in *V. natriegens*. Fluorescence is normalized to OD600 and presented here on a logarithmic scale. The course of fluorescence is presented for different inducer concentrations and at different time points.

##### 2.1.1.3. pMAN in *V. natriegens*

The mannose inducible promoter is part of the pJOE8889 plasmid and controls the expression of the Cas9 enzyme. Since we designed all CRISPR components to work with this plasmid we also had to make sure that Cas9 would be expressed upon addition of mannose. There are no reports in the literature on the use of this promoter so we characterized it with our reporter mCherry. Moreover, *V. natriegens* cannot utilize mannose as a carbon source. Addition of 1 (w/v) % led to a 2.5 fold increase of expression within 12 hours.

![Black and White QR](https://static.igem.org/mediawiki/2019/6/64/T--Groningen--Result19.png)

Figure 18 – pMAN is used in the pJOE8889 to control the expression of Cas9. In order to characterize this promoter we built it with mCherry and quantified the induction with mannose in *V. natriegens*.

#### 2.1.2 Constructs in *E. coli*

We characterized the pBad (BBa\_K808000), pTet (BBa\_R0040) and pMan (BBa\_J100303) promoter using mCherry as a reporter. For pBad we observe a 10 fold increase of mCherry expression incubating them for 8 hours with 1 % arabinose (figure 19). Induction with anhydrotetracycline for 8 hours let to a 20 times increase of mCherry fluorescence (figure 20). We also characterized the mannose inducible promoter which was constitutive in our measurements (figure 21).

![Black and White QR](https://static.igem.org/mediawiki/2019/b/b0/T--Groningen--Result20.png)

Figure 19 – pBAD is a commonly used promoter offering tight regulation of gene expression. Here, we characterize the promoter in *E. coli* using mCherry as reporter after the induction with arabinose.

![Black and White QR](https://static.igem.org/mediawiki/2019/3/3e/T--Groningen--Result21.png)

Figure 20 – We characterize the pTET promoter in *E. coli* using mCherry as reporter after the induction with anhydrotetracycline.

![Black and White QR](https://static.igem.org/mediawiki/2019/1/10/T--Groningen--Result22.png)

Figure 21 – pMAN is used in the pJOE8889 to control the expression of Cas9. In order to characterize this promoter we built it with mCherry and quantified the induction with mannose in *E.coli*.

### 2.2 Synthetic promoter libraries

We engineered two synthetic promoter libraries with the aim to improve inducibility and function in *E. coli* and *V. natriegens*. We selected the arabinose (BBa\_K808000) and tetracycline (BBa\_R0040) as targets for our design following the recommendation from Tanya Tschirhart, who experienced a lower induction of both promoters in *V. natriegens*, compared to *E. coli*. In order to construct the library we followed the design from Denmark iGEM team 2010. We designed primers that would amplify the plasmid with the original pTET\_mCherry or pBAD\_mCherry construct and yield randomized sequences between the -35 and -10 region of the promoters. Additionally, the overlapping part of the promoter contains a restriction site that was used for circularization of the plasmid.

![Black and White QR](https://static.igem.org/mediawiki/2019/thumb/6/65/T--Groningen--Result23.png/1106px-T--Groningen--Result23.png)

The constructed plasmids were transformed into both *E. coli* and *V. natriegens* and appearing colonies were screened for inducible fluorescence with the corresponding inducer. In total 271 clones were measured with and without inducer, 245 for *E. coli* and 26 for *V. natriegens*. Numbers for *V. natriegens* are lower because of low transformation efficiency and the data are not shown. Data for all screened *E. coli* is displayed in the heat map, the first third of table representing data from pBAD, the rest being pTET. The presented numbers represent the fold change, that was achieved by dividing fluorescence in the presence of inducer by the fluorescence without it.

![Black and White QR](https://static.igem.org/mediawiki/2019/b/b8/T--Groningen--Result24.png)

Figure 22 – This heatmap gives an overview of the data collected for the SPL. The numbers represent the fold change increase in fluorescence after 8 h of growth with or without inducer. The first table represents the data measured for the SPL on pBAD. The other two tables represent the data measured for the SPL on pTET. The more intense the shade of red the higher the fold change.

This data allowed for the selection of 10 most promising clones. Again, fluorescence upon induction was measured, this time in duplicate. Most did not show an increase in fluorescence with the addition of the inducer. However, clone 6 from the batch induced with anhydrotetracycline reached a 34 times higher fluorescence in the presence of the inducer.

![Black and White QR](https://static.igem.org/mediawiki/2019/5/59/T--Groningen--Result25.png)

Figure 23: Clone 6 exhibits the highest fluorescence fold change upon induction. Single clones from the synthetic promoter library were induced with 250 ng/ml anhydrotetracycline. The fluorescence fold change is calculated by dividing the fluorescence in the induced state by fluorescence in the absence of inducer. The used fluorescence reporter is mCherry.

Apart from this strong inducibility we also checked the basal level of fluorescence. Comparison with the background fluorescence of *E. coli* reveals that indeed there is no leaky transcription of mCherry in absence of the inducer.

![Black and White QR](https://static.igem.org/mediawiki/2019/4/49/T--Groningen--Result26.png)

Figure 24: The basal level of reporter expression is comparable to *E. coli* without the construct. Clone 6 from the synthetic promoter library was induced with 250 ng/ml anhydrotetracycline. The used fluorescence reporter is mCherry.

We finally isolated and sequenced the plasmid to get the sequence of our optimized tetracycline inducible promoter.

![Black and White QR](https://static.igem.org/mediawiki/2019/8/85/T--Groningen--Result27.png)

### 2.3. CRISPR

Knock out using CRISPR applies the Cas9 enzyme, which with the help of a gRNA finds a target and introduces a double stranded break. If such a break happens within the genome of an organism, it has to be repaired to stay viable. One method to repair the break is using a homologous part as a template to ensure functionality of the repaired sequence. In genetic engineering, we customize both the gRNA, i.e. the breaking point, and the homologous part, i.e. the final sequence after the repair.

Our motivation to apply CRISPR was the knock out of the HisD enzyme, which is responsible for the last step of the histidine biosynthesis pathway. Missing functionality of this enzyme would lead to auxotrophy. Using a histidine auxotroph would require the addition of this amino acid if you want to read our message, adding another layer of security.

We designed three gRNA target sites using Synthego software and three homologous parts of different length complementary to the up and down stream region of HisD. A summary of all these parts can be found in our basic parts page. We cloned the gRNA into the pJOE8889 plasmid and transformed them along with the homologous parts. The transformations were first plated on medium plates containing the resistance marker kanamycin and the inducer mannose. Colonies that appeared within 2 days were plated on minimal medium with or without histidine to identify auxotrophy. Clones that would grow only if histidine is supplied are auxotrophic. However, this screening revealed that no auxotrophy was achieved so the protocol should be revised and optimized in the future.

##### Literature References:

[1] [Lehner,
B.A.E.; Schmieden, D.T.; Meyer, A.S. A Straightforward Approach for 3D Bacterial
Printing. ACS Synth Biol. doi: 10.1021/acssynbio.6b00395](https://pubs.acs.org/doi/10.1021/acssynbio.6b00395)
[2] [Kuo, C.K.; Ma, P.X. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials. doi: 10.1016/S0142-9612(00)00201-5](https://www.sciencedirect.com/science/article/pii/S0142961200002015)
[3] [Weinstock, M.T.; Hesek, E.D.; Wilson, C.M.; Gibson, D.G. Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods. doi: 10.1038/nmeth.3970](https://www.nature.com/articles/nmeth.3970)
[4] [Hammar, M.; Bian, Z.; Normark, S. Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.93.13.6562](https://www.pnas.org/content/93/13/6562.short)
[5] [Wang, X.; Smith, D.R.; Jones, J.W.; Chapman, M.R. In vitro polymerization of a functional Escherichia coli amyloid protein. J Biol Chem. doi: 10.1074/jbc.M609228200](http://www.jbc.org/content/282/6/3713.short)
[6] [Spiesz, E.M.; Yu, K.; Lehner, B.A.E.; Schmieden, D.T.; Aubin-Tam, M.E.; Meyer, A.S. Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter. J Vis Exp. doi: 10.3791/59477](https://www.jove.com/video/59477/three-dimensional-patterning-engineered-biofilms-with-do-it-yourself)

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