iGEM 2019 — Team Marburg

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

# Team:Marburg/Notebook

[![](https://static.igem.org/mediawiki/2019/3/3f/T--Marburg--header_logo.svg)](https://2019.igem.org/Team%3AMarburg)

Team

[Team Members](https://2019.igem.org/Team%3AMarburg/Team)
[Collaboration](https://2019.igem.org/Team%3AMarburg/Collaborations)
[Attribution](https://2019.igem.org/Team%3AMarburg/Attributions)
[Giant Jamboree](https://2019.igem.org/Team%3AMarburg/Giant_Jamboree)

Project

[Description](https://2019.igem.org/Team%3AMarburg/Description)
[Design](https://2019.igem.org/Team%3AMarburg/Design)
[Experiments](https://2019.igem.org/Team%3AMarburg/Experiments)
[Notebook](https://2019.igem.org/Team%3AMarburg/Notebook)
[Results](https://2019.igem.org/Team%3AMarburg/Results)
[Metabolic](https://2019.igem.org/Team%3AMarburg/Metabolic)
[Contribution](https://2019.igem.org/Team%3AMarburg/Contribution)
[Demonstrate](https://2019.igem.org/Team%3AMarburg/Demonstrate)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Safety](https://2019.igem.org/Team%3AMarburg/Safety)

Parts

[Parts Overview](https://2019.igem.org/Team%3AMarburg/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Improve](https://2019.igem.org/Team%3AMarburg/Improve)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)

[Judging Form](https://igem.org/2019_Judging_Form?team=Marburg)

Labautomation

[Story](https://2019.igem.org/Team%3AMarburg/Labautomation)
[Colony Picking](https://2019.igem.org/Team%3AMarburg/Colony_Picking)
[Cloning Protocols](https://2019.igem.org/Team%3AMarburg/Cloning_Protocols)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Miniprep](https://2019.igem.org/Team%3AMarburg/Miniprep)

Awards

[Integrated Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)
[Best New Basic Part](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Best New Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Best New Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Software](https://2019.igem.org/Team%3AMarburg/Software)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Best Plant Synthetic Biology](https://2019.igem.org/Team%3AMarburg/Plant)

[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)

# N O T E B O O K

---

![Syntex Logo](https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg)

M A R C H

|  |  |
| --- | --- |
| 7. | First talk with Pascal Pfister about Cyano project |
| 21. | Labfolder Seminar |
| 22. - 23. | Project planning weekend |
| 26. | Finished Syntex logo |
| 31. | First time in the lab with cyanos |

A P R I L

|  |  |
| --- | --- |
| 1. - 2. | Postertalk at Max-Planck-Institute for Terrestrial Microbiology Marburg |
| 1. - 7. | Project reveal on social media |
| 3. | Labsupply Frankfurt |
| 4. | PCC tryout transformation |

M A Y

|  |  |
| --- | --- |
| 2. | Skype meeting with Prof. Annegret Wilde |
| 3. - 5. | [iGEM Spring Festival in Bonn](https://2019.igem.org/Team%3AMarburg/Collaborations#meetups) |
| 4. | [March4Science in Frankfurt](https://2019.igem.org/Team%3AMarburg/Public_Engagement) |
| 14. | Promega Webinar |
| 17. - 19.9. | [iGEM Meetup in Erlangen](https://2019.igem.org/Team%3AMarburg/Collaborations#meetups) |
| 20. | Skype meeting with Lars Bähr (CEO) from CellDeg |
| 21. - 23. | Labvolution in Hannover |
| 28. | Conference call with Berthold Technologies Measurement Systems |

J U N E

|  |  |
| --- | --- |
| 3. | iGEM BBQ with students in Marburg  ---  Skype meeting with Licher |
| 4. - 6. | [Meetup with Prof. Annegret Wilde in Freiburg](https://2019.igem.org/Team%3AMarburg/Human_Practices#prof_wilde) |
| 8. - 9. | [Public Engagement at the Hessian Day in Bad Hersfeld](https://2019.igem.org/Team%3AMarburg/Public_Engagement) |
| 12. | Skype meeting with Darcia Schweitzer from Promega |
| 13. | iGEM BBQ with students in Marburg |
| 18. | Symposium:  *You never walk alone - We and our microbiome organized by Synmikro* |
| 23. | Teambuilding in the Kletterwald Marburg |
| 23. - 27. | Promega Instagram-Story takeover |
| 24. | Meeting with Kurt Weller from Sparkasse Frankfurt |

J U L Y

|  |  |
| --- | --- |
| 1. | [Skype meeting with the Delft iGEM team](https://2019.igem.org/Team%3AMarburg/Collaborations#delft) |
| 5. - 7. | [iGEM Meetup Düsseldorf](https://2019.igem.org/Team%3AMarburg/Collaborations#meetups) |
| 13. | Project planning |
| 17. | Meeting with the mayor:  Future of iGEM in Marburg |
| 23. | Skype meeting with iGEM team Aachen "Plastractor"  ---  [Skype meeting with iGEM team Delft](https://2019.igem.org/Team%3AMarburg/Collaborations#delft) |
| 30. | [First Golden Gate Webinar for European region](https://2019.igem.org/Team%3AMarburg/Collaborations#golden_gate) |
| 31. | [Second Golden Gate Webinar for Asian/American region](https://2019.igem.org/Team%3AMarburg/Collaborations#golden_gate) |

A U G U S T

|  |  |
| --- | --- |
| 1. - 7. | [Biohackathon Vilnius](https://2019.igem.org/Team%3AMarburg/Collaborations#meetups) |
| 9. | [Third Golden Gate Webinar for Asian/American region](https://2019.igem.org/Team%3AMarburg/Collaborations#golden_gate) |
| 14. | Donating blood with the team |
| 20. | Skype meeting with Dr. Dan Kramer (CEO) from [Cyano Biotech](https://2019.igem.org/Team%3AMarburg/Human_Practices#cyano_biotech)   ---  [Skype meeting with iGEM team Delft](https://2019.igem.org/Team%3AMarburg/Collaborations#delft) |
| 21. | Skype meeting with David De Lucrezia (managing director) from [Doulix](https://2019.igem.org/Team%3AMarburg/Human_Practices#doulix) |
| 28. | [Magarethe Schwarz from Promega visits the team](https://2019.igem.org/Team%3AMarburg/Human_Practices#promega)   ---  Skype meeting with [Nina Scheer from the Social Democratic Party of Germany (SPD)](https://2019.igem.org/Team%3AMarburg/Human_Practices#nina_scheer) |

S E P T E M B E R

|  |  |
| --- | --- |
| 10. | Announcing the [colony picture collaboration winners](https://2019.igem.org/Team%3AMarburg/Collaborations#colony_pictures) |
| 11. - 13. | [Cyano Meetup in Tübingen organized by VAAM](https://2019.igem.org/Team%3AMarburg/Human_Practices#cyano_conference) |
| 14. | [Skype meeting with iGEM team Berlin](https://2019.igem.org/Team%3AMarburg/Collaborations#berlin) |
| 14. - 15. | Public engagement during the plant market in the botanical garden of Marburg |
| 26. | [Interview for "Forschung aktuell in DLF"](https://www.deutschlandfunk.de/synthetische-biologie-wie-cyanobakterien-aus-sonnenlicht.676.de.html?dram:article_id=462031) by Michael Lange  ---  [Panel discussion about genetic engineering](https://2019.igem.org/Team%3AMarburg/Human_Practices#panel_discussion target=) organized by the team |

O C T O B E R

|  |  |
| --- | --- |
| 9. | Meeting with Dr. Friedhelm Nonne (Universities Chancellor) |
| 31. - 4.11. | Giant Jamboree in Boston |

![THM](https://static.igem.org/mediawiki/2019/f/f5/T--Marburg--logo_thm.svg)

![Marburg](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--logo_marburg.svg)

![Gießen](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--logo_jlu.svg)

![Mail](https://static.igem.org/mediawiki/2019/2/2c/T--Marburg--m_icon_ma.svg)
[![Instagram](https://static.igem.org/mediawiki/2019/e/e7/T--Marburg--m_icon_in.svg)](https://www.instagram.com/igem.marburg.2019)
[![Twitter](https://static.igem.org/mediawiki/2019/4/43/T--Marburg--m_icon_tw.svg)](https://twitter.com/igemmarburg2019)
[![Facbook](https://static.igem.org/mediawiki/2019/b/b1/T--Marburg--m_icon_fa.svg)](https://www.facebook.com/IGEMMarburg2019/)


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

# Team:Marburg/Experiments

[![](https://static.igem.org/mediawiki/2019/3/3f/T--Marburg--header_logo.svg)](https://2019.igem.org/Team%3AMarburg)

Team

[Team Members](https://2019.igem.org/Team%3AMarburg/Team)
[Collaboration](https://2019.igem.org/Team%3AMarburg/Collaborations)
[Attribution](https://2019.igem.org/Team%3AMarburg/Attributions)
[Giant Jamboree](https://2019.igem.org/Team%3AMarburg/Giant_Jamboree)

Project

[Description](https://2019.igem.org/Team%3AMarburg/Description)
[Design](https://2019.igem.org/Team%3AMarburg/Design)
[Experiments](https://2019.igem.org/Team%3AMarburg/Experiments)
[Notebook](https://2019.igem.org/Team%3AMarburg/Notebook)
[Results](https://2019.igem.org/Team%3AMarburg/Results)
[Metabolic](https://2019.igem.org/Team%3AMarburg/Metabolic)
[Contribution](https://2019.igem.org/Team%3AMarburg/Contribution)
[Demonstrate](https://2019.igem.org/Team%3AMarburg/Demonstrate)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Safety](https://2019.igem.org/Team%3AMarburg/Safety)

Parts

[Parts Overview](https://2019.igem.org/Team%3AMarburg/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Improve](https://2019.igem.org/Team%3AMarburg/Improve)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)

[Judging Form](https://igem.org/2019_Judging_Form?team=Marburg)

Labautomation

[Story](https://2019.igem.org/Team%3AMarburg/Labautomation)
[Colony Picking](https://2019.igem.org/Team%3AMarburg/Colony_Picking)
[Cloning Protocols](https://2019.igem.org/Team%3AMarburg/Cloning_Protocols)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Miniprep](https://2019.igem.org/Team%3AMarburg/Miniprep)

Awards

[Integrated Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)
[Best New Basic Part](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Best New Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Best New Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Software](https://2019.igem.org/Team%3AMarburg/Software)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Best Plant Synthetic Biology](https://2019.igem.org/Team%3AMarburg/Plant)

[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)

# E X P E R I M E N T S

---

![Syntex Logo](https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg)

*"When you're experimenting you have to try so many things before you choose what you want, and you may
go days getting nothing but exhaustion."*- **Fred Astaire**

# P R O T O C O L S

---

All the protocols used in our project are listed here.

# Protocols

X

## Cultivation

[ ]

BG media

---

**5x BGM media:**

Stock 1 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| Na2Mg EDTA | 4.839 g/L |
| Ferric ammonium citrate | 6.287 g/L |
| CaCl2 x 2 H2O | 17.936 g/L |
| citric acid x 1 H2O | 2.997 g/L |

Stock 2 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| MgSO4 x 7 H2O | 37.464 g/L |

Stock 3 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| K2HPO4 | 10.451 g/L |
| KH2PO4 | 8.165 g/L |

Stock 4 (500x microelements): autoclave

|  |  |
| --- | --- |
| H3BO3 | 7.139 g/L |
| MnCl2 x 4 H2O | 4.522 g/L |
| ZnSO4 x 7 H2O | 311.599 mg/L |
| CuSO4 x 5 H2O | 126.092 mg/L |
| NaMoO4 | 122.233 mg/L |
| CoCl2 x 6 H2O <- solve first in 30 ml water and add to stock 4. | 975.059 mg/L |

Combine stock solutions and add from **1 M HEPES/NaOH buffer (pH 8.0)** stock to a final
concentration of **20 mM**.

Add **8.159 g of NaNO3 per liter** of medium.

For solid media add **1.4 % Agar**.

Autoclave.
After autoclaving add filter sterilized **Na2S2O3** to a final
concentration of **1 mM** to 5x BGM-Agar.

This recipe originated from the publication [Włodarczyk *et. al.*, 2019](https://doi.org/10.1101/684944) and has been adapted by the iGEM Team Marburg 2019.

**BG11 medium from Uni Marburg**

Stock 1 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| Na2Mg EDTA | 0.1 g/L |
| Ferric ammonium citrate | 0.6 g/L |
| CaCl2 x 2 H2O | 3.6 g/L |
| citric acid x 1 H2O | 0.6 g/L |

Stock 2 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| MgSO4 x 7 H2O | 7.5 g/L |

Stock 3 (100x): filter sterilize or autoclave

|  |  |
| --- | --- |
| K2HPO4  or K2HPO4 x 3 H2O | 3.05 g/L 4.0 g/L |

Stock 4 (1000x microelements): autoclave

|  |  |
| --- | --- |
| H3BO3 | 2.88 g/L |
| MnCl2 x 4 H2O | 1.81 g/L |
| ZnSO4 x 7 H2O | 0.222 g/L |
| CuSO4 x 5 H2O | 0.079 g/L |
| CoCl2 x 6 H2O | 0.05 g/L |
| NaMoO4 | 0.391 g/L |

Combine stock solutions and add from **1 M HEPES/NaOH buffer (pH 8.0)** stock to a final
concentration of **20 mM**.

Add **1.5 g of NaNO3 per liter** of medium.

For solid media add **1.4 % Agar**.

Autoclave.

After autoclaving add filter sterilized **Na2S2O3** to a final
concentration of **1 mM** to BG11-Agar.

**BG11 medium from Düsseldorf**

Final concentration of medium:

Compound | Concentration || CaCl2 x 2 H2O | 0.036 g/L |
| citric acid | 0.0006 g/L |
| NaNO3 | 1.4958 g/L |
| MgSO4 x 7 H2O | 0.0749 g/L |
| 0.25 M Na2EDTA (pH 8) | 0.0056 mL/L |
| Na2CO3 | 20 µg/L |
| Fe(III) ammonium citrate | 6 µg/L |
| K2HPO4 x 3 H2O | 30 µg/L |
| HEPES (pH 8) | 10 mM |
| H3BO3 | 2.86 mg/L |
| MnCl2 x 4 H2O | 1.81 mg/L |
| ZnSO4 x 7 H2O | 0.222 mg/L |
| Na2MoO4 x 2 H2O | 0.390 mg/L |
| Co(NO3)2 x 6 H2O | 0.049 mg/L |
| (CuSO4 x 5 H2O | 0.079 mg/L if required) |

Guidelines:
Always work under sterile conditions when handling sterile media or stocks.
Work
under the clean bench.

Safety warnings:
Wear gloves when preparing stocks!
Heavy metals are toxic for the
environment and need to be discarded accordingly.

Before starting:
For plates: Thaw antibiotic stocks before pouring plates.

1. 100x BG11 stock:

   |  |  |
   | --- | --- |
   | CaCl2 x 2 H2O | 3.6 g/L |
   | citric acid | 0.6 g/L |
   | NaNO3 | 149.58 g/L |
   | MgSO4 x 7 H2O | 7.49 g/L |
   | 0.25 M Na2EDTA, pH 8 | 0.56 mL/L |

   For 100x BG11 Stock-N:
   Omit NaNO3

2. Supplemental stocks for standard media:

   |  |  |
   | --- | --- |
   | 1000x Na2CO3 | 20 mg/mL |
   | 100x TES-buffer, pH 8.0 (1M) | adjust with KOH |
   | 1000x K2HPO4 x 3 H2O | 30 mg/mL |
   | 1000x Fe(III) ammonium citrate | 6 mg/mL |
   | 5000x CuSO4 x 5 H2O | 395 ng/mL |

3. Trace metal mix (1000x concentration):

   |  |  |
   | --- | --- |
   | H3BO3 | 2.86 g/L |
   | MnCl2 x 4 H2O | 1.81 g/L |
   | ZnSO4 x 7 H2O | 0.222 g/L |
   | Na2MoO4 x 2 H2O | 0.390 g/L |
   | Co(NO3)2 x 6 H2O | 0.049 g/L |

   For BG11 lacking certain metals (e.g. for working with metal inducible promoters
   P*petE*, P*coaT*, P*ziaA* etc., trace
   metal mix can be prepared lacking these chemicals and used instead of standard trace metal mix.

4. Standard 1x BG11:

   Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.

   | Stock solution | Volume |
   | --- | --- |
   | 100x BG11 Stock | 10 mL |
   | 1000x Na2CO3 | 1 mL |
   | 1000x K2HPO4 x 3 H2O 100x TES-buffer | 1 mL 10 mL |
   | 100x TES-buffer | 10 mL |
   | 1000x Trace metal Mix | 1 mL |

   Add ultra pure water to 1 L.
   Autoclave.
   After autoclaving, add 1 mL 1000x Fe(III)
   ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x CuSO4

5. Standard 1x BG11-N:

   Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.

   | Stock solution | volume |
   | --- | --- |
   | 100x BG11 Stock-N | 10 mL |
   | 1000x Na2CO3 | 1 mL |
   | 1000x K2HPO4 x 3 H2O 100x TES-buffer | 1 mL 10 mL |
   | 100x TES-buffer | 10 mL |
   | 1000x Trace metal Mix | 1 mL |

   Add ultra pure water to 1 L.
   Autoclave.
   After autoclaving, add 1 mL sterile 1000x
   Fe(III) ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x
   CuSO4

6. Standard 2x BG11 for agar plates:

   Fill 500 mL bottle with 250 mL ultra pure water. Add stock solutions as shown below.

   | Stock solution | Volume |
   | --- | --- |
   | 100x BG11 Stock-N | 10 mL |
   | 1000x Na2CO3 | 1 mL |
   | 1000x K2HPO4 x 3 H2O 100x TES-buffer | 1 mL 10 mL |
   | 100x TES-buffer, pH = 8.0 | 10 mL |
   | 1000x Trace metal Mix | 1 mL |

   Add ultra pure water to 500 mL.
   Autoclave.
   After autoclaving, add 1 mL sterile
   1000x Fe(III) ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x
   CuSO4

7. BG11 plates:

   Prepare 1.5 % agar: Weigh 4.5 g Bacto Agar. Fill up to 300 mL. Autoclave.

   Microwave agar until liquid. Let cool.

   In a 50 mL Falcon, add 1 vol 2x BG11 and 1 vol liquid 1.5 % agar. (Note: Usually, one
   plate requires 30-40 mL total volume.)

   When mixture is hand warm, add appropriate antibiotics, if required. Quickly pour late,
   avoiding air bubbles.

**BG11 medium from Tübingen**

For 1 L *n* mL of the stock solutions are used:
stock solution 1-7: 5 mL
stock
solutin 8: 5 mL
stock solution of trace elements: 1 mL

| BG11-media stock solutions (200x) | m (for 200 mL) | final concentration |
| --- | --- | --- |
| 1.   NaNO3 | 60.00 g | 17.65 mM |
| 2.   K2HPO4 | 1.25 g | 0.18 mM |
| 3.   MgSO4 x 7 H2O | 2.96 g | 0.30 mM |
| 4.   CaCl2 x 2 H2O | 1.47 g | 0.25 mM |
| 5.   Na2EDTA 2 x H2O | 0.04 g | 0.003 mM |
| 6.   Na2CO3 | 1.61 g | 0.38 mM |
| 7.   Fe(III) citrate citric acid | 0.29 g 0.23 g | 0.03 mM 0.03 mM |
| 8.   NaHCO3 | 16.80 g | 5 mM |

| Trace elements stock solution (1000x) | m (for 100 mL) | final concentration |
| --- | --- | --- |
| H3BO4 | 286 mg | 46.26 µM |
| MnCl2x 4 H2O | 181 mg | 9.15 µM |
| ZnSO4 x 7 H2O | 22.2 mg | 0.77 µM |
| Na2MO4 x 2 H2O | 39 mg | 1.61 µM |
| CuSO4 5 x H2O | 7.9 mg | 0.32 µM |
| Co(NO3)2 x 6 H2O | 4.94 mg | 0.17 µM |

**Preparation of stock solutions:**

stock solutions 1-7: weigh out substances and fill up with 200 mL
MilliQ-H2O.
Note:
Fe(III) citrate and citric acid are weighed in together and have to be protected from
light.
Autoclave stock solutions and store them at room temperature. Stock solution 7
should
be
wrapped up in aluminium foil.

**Preparation of BG11-medium:**

give 900 mL MilliQ H2O in a 1 L measuring cylinder.
5 mL of each 200x stock
solution are added (7 stock solutions)
1 mL trace element stock solution (1000x) is added.

Fill the cylinder with MilliQ H2 O up to 1000 mL or 995 mL.
Autoclave.
There
will be a precipitate so shake before use.
Store at room temperature.
Add 5 mL 1 M
NaHCO3 stock solution (8) (200x) before use.

**agar plates:**

end concentration 10 g/L agar (Use Bacto-Agar for cyanobacteria!)
Mix agar and
BG11-medium
after autoclaving.
For 1L agar plates: Autoclave 10 g agar in 500 mL MilliQ-H2O
and
500 mL 2x BG11 (5 mL of each stock solution + 1 mL of the trace element stock solution)
seperately.
Mix them when handwarm and add 5 mL NaHCO3 (+ Glc +
Casaminoacids)
500 mL last for approximately one bag of petri dishes.
Pour thick agar
plates (30-40 mL) as cyanobacteria have long generation times.
**Verify that there are no
contaminations: (Rippka et al., 1979)**
BG11 agar plates + 2 % glucose + 0.02 %
Casaminoacids (for 500 mL agar plates):
20 mL glucose (50 % stock solution, 55 g glucose
monohydrate on 100 mL (warm up to dissolve) --> sterile filtration)
1 mL casaminoacids
(100
g/L stock solution).

[ ]

Competent cells

---

Competent cells (*E. coli*)

1. HK was inoculated with 2.00 mL of the overnight culture
2. Incubation until the optical density is 0.6 – 0.8 (37°C, 220 rpm)
3. Transfer into 50 mL falcons and centrifugation (5000 rpm, 10 min)
4. Remove supernatant
5. Resuspension in cold 0.1 M CaCl2 (15.0 mL)
6. Incubation on ice for 30 min
7. Centrifugation (5000 rpm, 10 min)
8. Remove supernatant
9. Resuspension in 0.1 M CaCl2 (3.25 mL) + 80% glycerin (0.75 mL)
10. Aliquot in sterile eppies
11. Blast-freeze in N2 (l)
12. Store in freezer (-80°C)

[ ]

Transformation

---

Transformation

1. x µL of DNA in either top10 or DH5α
   (x = 1.00 µL for Retransformation; 5.00 µL of GG
   lvl
   1 construct)
2. incubation on ice for 30 minutes
3. heatshock (42°C, 90 sec)
4. incubation in ice for 5 minutes
5. addition of 500 µL LB medium
6. incubation (37°C, x h)
   (x = 1 h when antibiotic resistance kanamycin, chloramphenicol,
   ampicillin; 2 h when spectinomycin)
7. centrifugation (5000 rpm, 2 min)
   (when doing a retransformation this step was not
   executed, 100 µL of liquid were then plated on an agar plate with antibiotic resistance)
8. remove supernatant
9. plate on agar plate with antibiotic resistance
10. incubation (37°C, overnight)

[ ]

Transformation protocol for Synechococcus 7942

---

Transformation protocol for Synechococcus 7942

1. Start a fresh culture (50 mL) in BG-11
2. Measure OD730nm to use cells when the OD is between 0.5 and 1.0
3. Spin down cells in sterile tube at 6000 × g at room temperature.
4. Concentrate the cells by re-suspending them in smaller amount of fresh growth medium so
   to
   get
   a final OD730nm of 2.5 – 3.5
5. Place 0.4 mL of re-suspended cells in sterile culture tubes.
6. Add 50 ng – 2 µg plasmid DNA to each tube and gently mix. (Leave one tube as a control
   w/o
   DNA
   added).
7. Wrap tubes in aluminum foil (perhaps make hole in Eppie lid for gas exchange).
8. Place the tubes in the growth chamber at 30° (if high CO2 requiring phenotype is
   expected
   place plates in 3 % CO2) for 4 – 24 h.
9. Spot 200 μL (about 10-15 μL drops) on a sterile filter (Whatman Nuclepore Track-Etch
   Membrane
   #111107, 47mm diameter; 0.4 μM pore size) that has been placed on a BG-11 agar plate +
   antibiotics.

[ ]

Triparental conjugation

---

Triparental conjugation

* UTEX 2973 culture was inoculated at OD 0.1
* When UTEX 2973 culture was at OD ~0.3, cultures of prK2013 and HB101 were inoculated
  from
  “overnight cultures” to OD 0.1
* When UTEX 2973 culture was at OD 0.6-0.8 the conjugation was started:

1. Centrifugation of 2.00 mL of each culture (4000 x g, 2min)
2. Washing (2x) with 2.00 mL of respective media
3. Pipet-mixing and inverting (no vortexing!)
4. Resuspension in media (UTEX 2973: 200 µL; E.coli: 100 µL)
5. Mixing of all three strains
6. Incubation (37°C, 30 minutes, 100-150 µE)
7. Blotting 5 µL on sterile filters on LB/BG11 (95:5) plates without antibiotics
8. Incubation (37°C, overnight, 150 µE)
9. Moving sterile filters on LB/BG11 (95:5) plates containing spectinomycin
10. Incubation (37°C, few days, 150 µE)
11. Streaking cyano blots on new BG11 plates containing spectinomycin
12. Colony PCR to verify the conjugation

[ ]

Well-plate cultivation

---

Well-plate cultivation

1. inoculate colony of UDAR 4787 into liquid media in Volume of 1 mL + 0.5 μL Spec.
   incubate
   (24-48h, 42 ˙C, 130 rpm, 5% CO2, 500 μE)
2. inoculate row A from 24-wellplate with preculture to OD730= 0.1
3. inoculate B6 with UDAR as blank
4. when OD reaches 0.6 inoculate row A to row C and D to OD = 0.1.
5. well B6 to well B4+5

## Measurement

[ ]

NanoLuc measurement

---

NanoLuc measurement

1. Inoculation of *E. coli* cultures (~0.1 OD 600 nm)
2. Let culture grow until OD ~ 0.8.
3. Preparation of Nano-Glo: mixing Nano-Glo buffer with NanoLuc substrate (1:50)
4. Add 50 µL of culture and 50 µL of substrate per well (96 well plate inoculation).
5. Wait 3 minuntes.
6. Measure luminescence

[ ]

Parts measurement

---

Parts measurement

after the well-plate cultivation (see above) the part measurement follows

1. when row C and D reach OD = 0.6 measure OD with plate reader
   Settings: 730 nm, 3
   measuring
   points (circle)
2. transfer 200 μL of blanks, C1-6 and D1-6 into 96 well plate black
3. measure fluorescence in a plate reader
4. Setting: Excitation 488 nm, Emission 518 nm, size 2x2 (circle), frame 1200 μm,
   strengthener:optimal

## Cloning

[ ]

Colony PCR protocol for *Synechococcus elongatus* UTEX 2973

---

Colony PCR protocol for *Synechococcus elongatus* UTEX 2973

1. Pick colony you want to use as template
2. Resuspend in 50µl ddH2O
3. Boil at 100°C for 10 minutes
4. Afterwards prepare the reaction as follows:

* 2.5 µl forward primer
* 2.5 µl reversed primer
* 25 µl Thermo Scientific™DreamTaq Green PCR Master Mix (2X)
* 20 µl boiled cells

5. Perform PCR using the following conditions:

| step | Temperature | Time | cycle number |
| --- | --- | --- | --- |
| Initial Denaturation | 95°C | 180 s | 1 |
| Denaturation | 95 | 30 s | 25 |
| Annealing | Tm | 30 s | 25 |
| Elongation | 72 | 60 s | 25 |
| Terminal elongation | 72 | 15 min | 1 |

For PCR products longer than 2kb, the elongation time should be prolonged by 1min/kb.
6. Load your gel

[ ]

Digestion

---

Digestion

1. measurement of concentration of DNA
2. mix components listed in table below

| component | volume /µL |
| --- | --- |
| DNA (1 µg) | x |
| H2O | 44.0-x |
| CutSmart Buffer or NEBuffer | 5.00 |
| Enzyme e.g. BsmbI or BSaI | 1.00 |
|  |  |
| --- | --- |
| Σ in tube | 50.0 |

3. 2h, 37°C in Mastercycler
4. Agarose gel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V,
   30-60
   minutes

[ ]

DNA plasmid purification with the Macherey-Nagel kit

---

DNA plasmid purification with the Macherey-Nagel Kit:

Before starting:

Add 1 mL of Buffer A1 to the RNase A vial and vortex. Transfer the solution back into the
Buffer
A1 bottle and mix thoroughly. Indicate date of RNase A addition. Store Buffer A1 containing
RNase
A at 4 °C. The solution will be stable at this temperature for at least six months.

Add the indicated volume of 96-100 % ethanol to Buffer A4 and Buffer AQ.

1. **Cultivate and harvest bacterial cells**

   Use **1-5 mL** of a saturated ***E.coli* LB culture**, pellet cells in a
   standard
   benchtop microcentrifuge for **30 s** at **11,000 x g**. Discard the supernatant and
   remove as much of the liquid as possible.

   *Note: For isolation of low-copy plasmids refer to section 5.2.*

   ~Personal note: We centrifuged in 15 mL falcons at 4000 rpm for 10 minutes at 4 °C.~

2. **Cell lysis**

   Add **250 µL Buffer A1**. Resuspend the cell pellet completely by vortexing or
   pipetting
   up and down. Make sure no cell clumps remain before addition of Buffer A2!

   *Attention: Check Buffer A2 for precipitated SDS prior to use. If a white
   precipitate is visible, warm the buffer several minutes at 30-40 °C until precipitate is
   dissolved completely. mix thoroughly and cool buffer down to room temperature (18-25
   °C).*

   Add **250 µL Buffer A2**. Mix gently by inverting the tube **6-8 times**. Do not
   vortex to avoid shearing of genomic DNA. incubate at **room temperature** for up to
   **5
   min** or until lysate appears clear.

   Add **300 µL Buffer A3**. Mix thoroughly by inverting the tube **6-8 times** until
   blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA!

   *Make sure to neutralize completely to precipitate all protein and chromosomal DNA.
   LyseControl should turn completely colorless without any traces of blue.*

3. **Clarification of lysate**

   Centrifuge for **5 min** at **11,000 x g** at room temperature.

   Repeat this step in case the supernatant is not clear!

4. **Wash silica membrane**

   Place a NucleoSpin® Plasmid / Plasmid (NoLid) Column in a Collection Tube (2 mL) and
   decant
   the supernatant from step 3 or pipette a maximum of 750 µL of the supernatant onto the
   column.
   Centrifuge for **1 min** at **11,000 x g**. Discard flowthrough and place the
   NucleoSpin® Plasmid / Plasmid (NoLid) Column back into the collection tube.

   Repeat this step to load the remaining lysate.

5. **Wash silica membrane**

   *Recommended: If plasmid DNA is prepared from host strains containing high levels of
   nucleases (e.g., HB101 or strains of the JM series), **it is strongly recommended**
   performing an additional washing step with **500 µL Buffer AW, optionally preheated to
   50
   °C**, and centrifuge for **1 min** at **11,000 x g** before proceeding with
   Buffer
   A4. Additional washing with Buffer AW will also increase the reading length of DNA
   sequencing reactions and improve the performance of critical enzymatic reactions.*

   Add **600 µL Buffer A4** (supplemented with ethanol, see section 3). Centrifuge for
   **1
   min** at **11,000 x g**. Discard flowthrough and place the NucleoSpin®
   Plasmid/Plasmid
   (NoLid) Column back into the **empty** collection tube.

6. **Dry silica membrane**

   Centrifuge for **2 min** at **11,000 x g** and discard the collection tube.

   *Note: Residual ethanolic wash buffer might inhibit enzymatic reactions.*

7. **Elute DNA**

   Place the NucleoSpin® Plasmid/Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube
   (not
   provided) and add **50 µL Buffer AE**. Incubate for **1 min** at **room
   temperature**. Centrifuge for 1 min at room temperature. Centrifuge for 1 min at
   **11,000
   x g**.

   *Note: For more efficient elution procedures and alternative elution buffer
   (e.g.,
   TE buffer or water) see section 2.5.*

   ~Personal note: We heated Buffer AE to 80 °C and used 30-40 µL of it.~

[ ]

DpnI digest

---

DpnI digest

1. add 2 µL of DpnI to sample
2. incubate at 37°C for 2h
3. Agarosegel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V, 30-60
   minutes

[ ]

Gel Elektrophoresis

---

Gel Elektrophoresis

1. Preparation of the buffer and agarose gel

**TAE-buffer (50x):**

| Tris | EDTA-Na2-salt | acetic acid |
| --- | --- | --- |
| 242.3 g/L | 18.6 g/L | 60.05 g/L |

**1.2 % agarose gel:**

4.8 g agarose in 400 mL TAE-buffer (1x)

2. On 100 mL 1.2 % Agarose gel 5 µL EtBr are added, stirred, filled into the box and then let
   it
   solidify.
3. Add 10 µL of loading dye to 50 µL of the sample.
4. Fill the pockets of the gel: 3 µL of gene ladder in one pocket and 15-20 µL of sample in
   the
   other pockets.
5. Apply a voltage of 70-100 V and let gel run for 30-60 minutes.

[ ]

Gel extraction with the QIAquick® Gel Extraction kit

---

Gel extraction with the QIAquick® Gel Extraction kit

Notes before starting

* This protocoll is for the purification of up to 10 µg DNA (70 bp to 10 kb).
* The yellow colour of Buffer QG indicates a pH = 7.5. DNA adsorption to the membrane is
  only
  efficient at pH ≤ 7.5.
* Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
* Isopropanol (100%) and a heating block or water bath at 50°C are required.
* All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional
  table-top centrifuge.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colourless tube. Add 3 volumes of Buffer QG to 1 volume gel (100
   mg
   gel ~ 100 µL). The maximum amount of gel per spin column is 400 mg. For > 2% agarose gels,
   add
   6
   volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the
   tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check
   that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If
   the
   colour of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix.
   The
   mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 mL collection tube or into a vacuum
   manifold.
   To
   bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum
   to
   the manifold until all the samples have passed through the column. Discard the flow-through
   and
   place the QIAquick column back into the same tube. For sample volumes of >800 µL, load and
   spin/
   apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or
   microinjection,
   add 500 µL Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum.
   Discard
   flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 µL Buffer PE to QIAquick column and centrifuge for 1 min or apply
   vacuum.
   Discard flow-through and place the QIAquick column back into the same tube.
   **Note:** If the DNA will be used for salt-sensitive applications (e.g., sequencing,
   blunt-ended ligation), let the column stand 2-5 min after addition of Buffer PE.
   Centrifuge the QIAquick column in the provided 2 mL collection tube for 1 min to remove
   residual wash buffer.
8. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
9. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the centre of the
   QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add
   30
   µL Buffer EB to the centre of the QIAquick membrane, let the column stand for 1 min, and
   then
   centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing
   the
   incubation time up to 4 min can increase the yield of purified DNA.
10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of
    purified DNA. Mix the solution by pipetting up and down before loading the gel.

[ ]

Golden Gate level 0

---

Golden Gate level 0

1. Mix components listed in table below

| component | volume /µL |
| --- | --- |
| Entry vector (60 ng/µL) | 1.00 |
| TF buffer | 1.00 |
| T4 ligase | 1.00 |
| BsmbI | 1.00 |
| Insert (180 ng/µL) | x |
| H2O | 6.00-x |
|  |  |
| --- | --- |
| Σ in tube | 10.0 |

2. Golden Gate program in Mastercycler (Eppendorf)

Note: all the parts, the buffer and enzymes were kept on ice consistently

GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition
(30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C,
hold
until lid is opened

The protocol Golden Gate level 0 can also be found following [this link](https://www.protocols.io/view/golden-gate-lvl-0-8edhta6).

[ ]

Golden Gate level 1

---

Golden Gate level 1

1. Measurement of concentration of parts used
2. Dilution of parts to 20 fmol
3. Preparation of Mastermix (consisting of parts) in PCR-tube (1.00 µL of each part)
4. Addition of 1.00 µL level 1 Ori part
5. Addition of 1.00 µL T4 Lig buffer
6. Addition of 1.00 µL T4 Ligase
7. Addition of 1.00 µL BsaI
8. Pipet-mixing
9. GoldenGate program in Mastercycler (Eppendorf)

Note: all the parts, the buffer and enzymes were kept on ice consistently

GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition
(30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C,
hold
until lid is opened
GoldenGate spaceholder constructs program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min

Repetition (50x) of step 1 and 2
Step 3: 4°C, hold until lid is opened

The protocol Golden Gate level 1 can also be found following [this link](https://www.protocols.io/view/golden-gate-lvl-1-2-8d4hs8w).

[ ]

Ligation Protocol with T4 DNA Ligase (M0202) (New England BioLabs Inc.)

---

Ligation Protocol with T4 DNA Ligase (M0202) (New England BioLabs Inc.)

1. Set up the following reaction in a microcentrifuge tube on ice.*(T4 DNA Ligase should be
   added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to
   insert
   for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios*

| component | 20 µL reaction |
| --- | --- |
| T4 DNA Ligase Buffer (10X)\* | 2 µL |
| Vector DNA (4 kb) | 50 ng (0.020 pmol) |
| Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
| Nuclease-free water | to 20 μl |
| T4 DNA Ligase | 1 µL |

*\* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.*

2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature
   for
   2
   hours *(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute
   ligation).*
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

[ ]

PCR Purification with the QIAquick® PCR Purification Kit

---

PCR Purification with the QIAquick® PCR Purification Kit

Notes before starting

* This protocol is for the purification of up to 10 µg PCR products (100 bp to 10 kb in
  size).
* Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
* All centrifugation steps are carried out at 17.900 x g (13,000 rpm) in a conventional
  table-top microcentrifuge at room temperature.
* Add 1:250 volume pH indicator I to Buffer PB. The yellow colour of Buffer PB with pH
  indicator I indicates a pH ≤ 7.5. The adsorption of DNA to the membrane is only efficient at
  pH
  ≤ 7.5. If the purified PCR product is to be used in sensitive microarray applications, it
  may
  be
  beneficial to use Buffer PB without the addition of pH indicator I; do not add pH indicator
  I
  to
  buffer aliquots.
* Symbols: ● centrifuge processing; ▴ vacuum processing

1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the colour of the
   mixture
   is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The colour of the
   mixture
   will turn yellow.
2. Place a QIAquick column in ● a provided 2 mL collection tube or into▴a vacuum manifold.
   For
   details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.
3. To bind DNA, apply the sample to the QIAquick column and ● centrifuge for 30-60 s
   or▴apply
   vacuum to the manifold until all the samples have passed through the column. ● Discard
   flow-through and place the QIAquick column back in the same tube.
4. To wash, add 750 µL Buffer PE to the QIAquick column ● centrifuge for 30-60 s or▴apply
   vacuum. ● Discard flow-through and place the QIAquick column back in the same tube.
5. Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min
   to
   remove residual wash buffer.
6. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
7. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the
   centre
   of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA
   concentration,
   add 30 µL elution buffer to the centre of the QIAquick membrane, let the column stand for 1
   min
   and then centrifuge.
8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes
   of
   purified DNA. Mix the solution by pipetting up and down before loading the gel.

[ ]

PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

---

PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

1. Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols
   with
   other polymerases. Conditions recommended below should be used for optimal
   performance.
   **Reaction Setup:**
   We recommend assembling all reaction components on
   ice
   and quickly transferring the reactions to a thermocycler preheated to the denaturation
   temperature (98°C). All components should be mixed prior to use. Q5 High-Fidelity DNA
   Polymerase
   may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting
   errors.

| component | 25 µL reaction | 50 µL reaction | final concentration |
| --- | --- | --- | --- |
| 5X Q5 reaction Buffer | 5 µL | 10 µL | 1X |
| 10 mM dNTPs | 0.5 µL | 1 µL | 200 µM |
| 10 µM Forward Primer | 1.25 µL | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 1.25 μl | 2.5 µL | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Q5 High-Fidelity DNA Polymerase | 0.25 µL | 0.5 µL | 0.02 U/µL |
| 5X Q5 High GC Enhancer (optional) | (5 µL) | (10 µL) | 1X |
| Nuclease-Free Water | to 25 μl | to 50 µL |  |

*Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick
spin
if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated
lid.*
Transfer PCR tubes to a PCR machine and begin
thermocycling.
**Thermocycling
Conditions for a Routine PCR:**

| step | temp | time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25-35 cycles | 98°C \*50-72°C 72°C | 5-10 seconds 10-30 seconds 20-30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4-10°C |  |

*\*Use of the NEBTm Calculator is highly recommended.*

2. **General Guidelines:**
   Template:Use of high quality, purified DNA templates greatly
   enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as
   follows:

| DNA | amount |
| --- | --- |
| DNA Genomic | 1 ng - 1 µg |
| Plasmid or Viral | 1 pg - 10 ng |

3. Primers:
   Oligonucleotide primers are generally 20–40 nucleotides in length and ideally
   have
   a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze
   primers. The best results are typically seen when using each primer at a final concentration
   of
   0.5 µM in the reaction.
4. Mg++ and additives:
   Mg++ concentration of 2.0 mM is optimal for
   most
   PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final
   concentration
   of 1X, the Q5 Reaction Buffer provides the optimal Mg++
   concentration.
   Amplification of some difficult targets, like GC-rich sequences, may be
   improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer
   and
   should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer
   when
   other conditions have failed.
5. Deoxynucleotides:
   The final concentration of dNTPs is typically 200 μM of each
   deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not
   recommended
   for use with uracil-containing primers or templates.
6. Q5 High-Fidelity DNA Polymerase concentration:
   We generally recommend using Q5
   High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl
   reaction).
   However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40
   units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not
   exceed
   2 units/50 μl reaction, especially for amplicons longer than 5 kb.
7. Buffers:
   The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the
   first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such
   as
   GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer
   can
   improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0
   mM
   Mg++ at the final (1X) concentration.
8. Denaturation:
   An initial denaturation of 30 seconds at 98°C is sufficient for most
   amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes)
   for
   templates that require it.
   During thermocycling, the denaturation step should be kept to
   a
   minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
9. Annealing:
   Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to
   be
   higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the
   annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step
   at
   3°C above the Tm of the lower Tm primer. A temperature gradient can
   also
   be used to optimize the annealing temperature for each primer pair.
   For high
   Tm
   primer pairs, two-step cycling without a separate annealing step can be used (see note 12).
10. Extension:
    The recommended extension temperature is 72°C. Extension times are generally
    20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb
    for
    simple templates (plasmid, *E. coli*, etc.) or complex templates < 1
    kb.
    Extension
    time
    can
    be
    increased
    to
    40
    seconds
    per
    kb
    for
    cDNA
    or
    long,
    complex
    templates,
    if
    necessary.

    A final extension of 2 minutes at 72°C is recommended.
11. Cycle number:
    Generally, 25–35 cycles yield sufficient product. For genomic amplicons,
    30-35 cycles are recommended.
12. 2-step PCR:
    When primers with annealing temperatures ≥ 72°C are used, a 2-step
    thermocycling protocol (combining annealing and extension into one step) is possible.
13. Amplification of long products:
    When amplifying products > 6 kb, it is often helpful to
    increase the extension time to 40–50 seconds/kb.
14. PCR product:
    The PCR products generated using Q5 High-Fidelity DNA Polymerase have
    blunt
    ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is
    preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA
    Polymerase
    will degrade any overhangs generated.
    Addition of an untemplated -dA can be done with
    *Taq* DNA Polymerase (NEB #M0267 ) or Klenow exo- (NEB #M0212 ).

[ ]

qPCR

---

qPCR

1. Mix the components listed below

| component | Volume |
| --- | --- |
| template | 1 µL |
| Primer-F | 1 µL |
| Primer-R | 1 µL |
| 2x master mix (provided) | 10 µL |
| H2O | 7 µL |
|  |  |
| --- | --- |
| total volume | 20 µL |

2. Start program in Mastercycler:

1. 50°C, 2 minutes

2. 2.95°C, 2 minutes

3. 95°C, 15 seconds

4. 60°C, 60 seconds

cycles: 40(cycle 2 to 4)

[ ]

Quick electroporation of *E. coli*

---

Quick electroporation of *E. coli*

This quick electroporation protocol was explained to us by Dr. Alberto Sánchez-Pascuala Jerez
from the research group of Prof. Dr. Tobias J. Erb in the Max-Planck-Institute for terrestrial
microbiology in Marburg. It was adapted by us and might not be exactly how Dr. Alberto
Sánchez-Pascuala Jerez does it.

**Optional:**

1. Restreak cells from glycerol stock on LB.
2. Inoculate a single colony in liquid LB.

**Standard:**

1. Inoculate your culture at **OD600=0.05** from an overnight culture
2. Grow the culture until an **OD600=0.5** is reached. *Note: If the culture
   reaches OD600= ~0.6 it can still be used, but reinoculating is recommended if higher
   values
   are reached.*
3. Directly put the culture on **ice** for 10-15 min.
4. Transfer the culture into a falcon and spin down in a cooled centrifuge at **2500 rpm,
   4°C
   for 10-15 min.***Heraeus™ Multifuge™ X1 is used for higher volumes.*
5. **Wash** the cells 2-3 times in dd H2O (or other sterile water).
6. Resuspend in 0.5-2.0 mL water, depending on how many aliquots you want and how big the
   pellet
   is.
7. Make 50 µL – 100 µL aliquots.
8. **Add DNA** to your aliquot on ice.
9. Transfer cell/ DNA mix **into** an **electroporation cuvette** on ice. *Gene
   Pulser®/MicroPulser™ Electroporation Cuvettes (green cap) from Bio-Rad are used.*
10. **Wipe the cuvette** with a paper towel to remove any liquid that might cause an arc
    and
    place it in the electroporation chamber.
11. **Electroporate** the sample, directly **add recovery medium** and **transfer**
    the
    cells **into a reaction tube**. The following settings are used: 2500 V, 25 µF, 200 Ω, 2
    mm.
    As medium 500 µL SOB medium are added.
12. **Incubate** the cells at 37°C and 250 rpm for 1h (amp resistance) or 2h (other
    resistances; kan, cam, spec..)
13. **Plate** and incubate at 37°C over night.

[ ]

Sequencing

---

Sequencing

| substrate | volume |
| --- | --- |
| Primer | 3.00 µL |
| DNA (500-1200 ng) + H2O | 12.0 µL |
| ∑ in eppie | 15.0 µL |

1. Add Primer, DNA (500-1200 ng) and water
2. Attach sequencing label on eppie
3. Register the label number online on Microsynth website
4. send registered eppies to Microsynth

# L A B B O O K S

---

Our labbook entrys.

# Labbooks

X

* [Cultivation of UTEX](https://static.igem.org/mediawiki/2019/9/92/T--marburg--cultivation_of_UTEX.pdf)
* [Light measurement](https://static.igem.org/mediawiki/2019/c/c9/T--marburg--labbook-_light_measurement.pdf)
* [Building placeholder constructs lvl 1](https://static.igem.org/mediawiki/2019/e/ea/T--marburg--labbook-_building_placeholder_constructs_lvl_1.pdf)
* [Level 1 assembly method](https://static.igem.org/mediawiki/2019/7/7c/T--marburg--labbook-_level_1_assembly_method.pdf)
* [Toolbox: Building parts](https://static.igem.org/mediawiki/2019/9/93/T--marburg--labbook-Toolbox-_Building_parts.pdf)
* [*Synechococcus elongatus* PCC 7942](https://static.igem.org/mediawiki/2019/c/cf/T--marburg--labbook-_Synechococcus_elongatus_PCC_7942.pdf)
* [Toolbox: General](https://static.igem.org/mediawiki/2019/2/2c/T--marburg--labbook-_Toolbox-_general.pdf)
* [Toolbox Marburg Collection 1.0](https://static.igem.org/mediawiki/2019/6/6d/T--marburg--labbook-_Toolbox_copy_Marburg_Collection_1.pdf)
* [Triparental conjugation](https://static.igem.org/mediawiki/2019/6/63/T--marburg--labbook-_triparental_conjugation.pdf)
* [aNSo integration into the genome](https://static.igem.org/mediawiki/2019/8/83/T--marburg--labbook-_aNSo_integration_into_the_genome.pdf)
* [Schönas lvl 1 GG](https://static.igem.org/mediawiki/2019/0/09/T--marburg--labbook-_Sch%C3%B6nas_lvl_1_GG.pdf)
* [pANS domestication](https://static.igem.org/mediawiki/2019/2/21/T--marburg--labbook-_pANS_domestication.pdf)
* [Natural Competence](https://static.igem.org/mediawiki/2019/7/71/T--marburg--labbook-_Natural_Competence.pdf)
* [Growth Curves](https://static.igem.org/mediawiki/2019/f/f1/T--marburg--labbook-_Growth_Curves.pdf)
* [Strain Engineering: General](https://static.igem.org/mediawiki/2019/5/5d/T--marburg--labbook-_Generell_Strain_Engineering.pdf)
* [Cpf1 system](https://static.igem.org/mediawiki/2019/a/a5/T--marburg--labbook-_Cpf1_system.pdf)
* [NanoLuc + YFP (codonoptimised)](https://static.igem.org/mediawiki/2019/4/49/T--marburg--labbook-_Nanoluc_%2B_YFP_%28codonoptimised%29.pdf)
* [Farnesen-Synthase](https://static.igem.org/mediawiki/2019/d/d6/T--marburg--labbook-_Farnesen-Synthase.pdf)
* [MetabolicEngineering: General](https://static.igem.org/mediawiki/2019/9/92/T--marburg--labbook-_General_Metabolic.pdf)
* [Metabolic Engineering: General](https://static.igem.org/mediawiki/2019/9/92/T--marburg--labbook-_General_Metabolic.pdf)
* [Metabolic Engineering](https://static.igem.org/mediawiki/2019/1/10/T--marburg--labbook-_Hinrik_learn_how_to_use_labfolder.pdf)
* [Limonen-Synthase](https://static.igem.org/mediawiki/2019/6/69/T--marburg--labbook-_Limonen-Synthase.pdf)
* [MEP-Pathway](https://static.igem.org/mediawiki/2019/1/1c/T--marburg--labbook-_MEP-Pathway.pdf)
* [New parts/ improve parts](https://static.igem.org/mediawiki/2019/a/a5/T--marburg--labbook-_New_parts_Improve_parts_new.pdf)
* [qPCR](https://static.igem.org/mediawiki/2019/2/27/T--marburg--labbook-_qPCR_new.pdf)

# A U T O M A T I O N P R O T O C O L S

---

The protocols of the Automation Lab can be found here.

# Automation Lab protcols

X

[ ]

Hardware

---

Follow [this
link](https://github.com/igemsoftware2019/iGemMarburg2019/tree/master/Hardware) to see all the hardware designs of the Automation Lab.

[ ]

Plasmid Purification

---

Follow [this
link](https://github.com/igemsoftware2019/iGemMarburg2019/tree/master/Opentrons_Plasmid_Purification_Protocols) to see the plasmid purification protocols of the Automation Lab.

[ ]

Nuclear Integration Site Model

---

Follow [this
link](https://github.com/igemsoftware2019/iGemMarburg2019/tree/master/models/artificial_Neutral_integration_Site_options_%28aNSo%29) to see the nuclear integration site model of the Automation Lab.

[ ]

Faster R-CNN Implementation

---

Follow [this
link](https://github.com/igemsoftware2019/iGemMarburg2019/tree/master/AI) to see the faster R-CNN implementation protocol of the Automation Lab.

![THM](https://static.igem.org/mediawiki/2019/f/f5/T--Marburg--logo_thm.svg)

![Marburg](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--logo_marburg.svg)

![Gießen](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--logo_jlu.svg)

![Mail](https://static.igem.org/mediawiki/2019/2/2c/T--Marburg--m_icon_ma.svg)
[![Instagram](https://static.igem.org/mediawiki/2019/e/e7/T--Marburg--m_icon_in.svg)](https://www.instagram.com/igem.marburg.2019)
[![Twitter](https://static.igem.org/mediawiki/2019/4/43/T--Marburg--m_icon_tw.svg)](https://twitter.com/igemmarburg2019)
[![Facbook](https://static.igem.org/mediawiki/2019/b/b1/T--Marburg--m_icon_fa.svg)](https://www.facebook.com/IGEMMarburg2019/)

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

# Team:Marburg/Results

[![](https://static.igem.org/mediawiki/2019/3/3f/T--Marburg--header_logo.svg)](https://2019.igem.org/Team%3AMarburg)

Team

[Team Members](https://2019.igem.org/Team%3AMarburg/Team)
[Collaboration](https://2019.igem.org/Team%3AMarburg/Collaborations)
[Attribution](https://2019.igem.org/Team%3AMarburg/Attributions)
[Giant Jamboree](https://2019.igem.org/Team%3AMarburg/Giant_Jamboree)

Project

[Description](https://2019.igem.org/Team%3AMarburg/Description)
[Design](https://2019.igem.org/Team%3AMarburg/Design)
[Experiments](https://2019.igem.org/Team%3AMarburg/Experiments)
[Notebook](https://2019.igem.org/Team%3AMarburg/Notebook)
[Results](https://2019.igem.org/Team%3AMarburg/Results)
[Metabolic](https://2019.igem.org/Team%3AMarburg/Metabolic)
[Contribution](https://2019.igem.org/Team%3AMarburg/Contribution)
[Demonstrate](https://2019.igem.org/Team%3AMarburg/Demonstrate)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Safety](https://2019.igem.org/Team%3AMarburg/Safety)

Parts

[Parts Overview](https://2019.igem.org/Team%3AMarburg/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Improve](https://2019.igem.org/Team%3AMarburg/Improve)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)

[Judging Form](https://igem.org/2019_Judging_Form?team=Marburg)

Labautomation

[Story](https://2019.igem.org/Team%3AMarburg/Labautomation)
[Colony Picking](https://2019.igem.org/Team%3AMarburg/Colony_Picking)
[Cloning Protocols](https://2019.igem.org/Team%3AMarburg/Cloning_Protocols)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Miniprep](https://2019.igem.org/Team%3AMarburg/Miniprep)

Awards

[Integrated Human Practices](https://2019.igem.org/Team%3AMarburg/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3AMarburg/Public_Engagement)
[Model](https://2019.igem.org/Team%3AMarburg/Model)
[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)
[Best New Basic Part](https://2019.igem.org/Team%3AMarburg/Basic_Part)
[Best New Composite Part](https://2019.igem.org/Team%3AMarburg/Composite_Part)
[Best New Part Collection](https://2019.igem.org/Team%3AMarburg/Part_Collection)
[Software](https://2019.igem.org/Team%3AMarburg/Software)
[Hardware](https://2019.igem.org/Team%3AMarburg/Hardware)
[Best Plant Synthetic Biology](https://2019.igem.org/Team%3AMarburg/Plant)

[Measurement](https://2019.igem.org/Team%3AMarburg/Measurement)

# R E S U L T S

---

![Syntex Logo](https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg)

#

The way to the results we demonstrate here was full of success and failure. Therefore, it was
necessary
to
compare and revise our theoretical plans with the practical work and the associated results. After
trying our
best to implement our plans, we would like to show you on this page that we have managed to realize
some
of
our
goals and are able to show some achievements for every sub-group.

---

# S T R A I N E N G I N E E R I N G

---

By genetic modification of *S. elongatus* UTEX 2973 we succeeded the transformation of
plasmids in UTEX
2973.

# Strain engineering

X

Natural competence

Reintroducing natural competence into our *S. elongatus* strain was an
important goal for us.
To
make sure
that
our *S. elongatus* UTEX 2973 actually holds the point mutation in the
*pilN* gene we
thought
it has,
we sequenced
this region - and the results showed the expected mutation
![sequencing results](https://static.igem.org/mediawiki/2019/e/ed/T--Marburg--Seq_UTEX-pilN-with-o_iGEM_1_650.png)

Fig.1: Sequencing results showing the point mutation in the *pilN*
gene.

As there were multiple methods at hand that we could use to get our strain
naturally
competent
again,
we tried
all those at hand, making sure we do everything we can to tackle this issue.

Although not our favorite method, we tried integrating an intact copy of the
*pilN* gene into
neutral site
II of
*S. elongatus* UTEX 2973 following the example of  [Li *et al.*, 2018](https://doi.org/10.1016/j.ymben.2018.06.002) .
We received pSII-trc-pilN,
the same plasmid used by Li *et al.*, as a gift from Petra Wurmser from the
research group of Prof.
Kaldenhoff in Darmstadt and conjugated it into our strain via triparental
conjugation.

![map](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--p_pilN-NSII.png)

Fig. 2: Plasmidmap of pSII-trc- *pilN* .

Beforehand, Petra Wurmser told us that she was not able to successfully
reproduce
the experiment,
motivating
us
to try it ourselves.
We made sure to follow the protocol in the above mentioned paper, transforming
pRL443 and pRL623
into *E.
coli*
HB101 and pSII-trc-pilN into *E. coli* HB101. Overnight cultures of these
cells
were inoculated and grown to
OD600≈0.5.
After washing and incubating them together for half an hour, they were mixed
with a
exponentially
growing
*S. elongatus* culture and incubated for 30 minutes again. Thereafter the
mixture was blotted
on
sterile filters and
incubated on BG11 plates for 24h before being transferred onto BG11 plates
containing kanamycin
 [(Li *et al.*,
2018)](https://doi.org/10.1016/j.ymben.2018.06.002).
It is important to add
that we also went with another approach for conjugation: Martina Carrillo
Camacho
from the working
group of Prof. Dr. Tobias Erb provided us with the pRK2013 plasmid, mentioning
that
she uses it
for conjugation. So we transformed the plasmid into  *E. coli*  DH5ɑ and
pSII-trc-pilN into
HB101, performing the
same
procedure with them as stated above.

After a whole week we could actually see growing colonies, though they were only
from attempts
with the
pRK2013 plasmid.

![map](https://static.igem.org/mediawiki/2019/e/e5/T--Marburg--Trafo-pilN-NSII-UDAR.jpg)

Fig.3 : Pictures of agarplates with colonies received from the triparental
conjugation with
the
helperstrain pRK2013 and HB101, haboring the pSII-trc-*pilN* vector.

We were still excited, directly starting liquid cultures and
running
colony PCRs in hope to find our desired result. Although trying a wide variety
of
primer
combinations,
we were
not able to find any successful integrations, but as the strain was growing on
kanamycin and
colony PCR
does not
always work correctly, we wanted to make sure and tried an actual
transformation: A
YFP construct
was
transformed
into our seemingly competent strain, allowing for easy selection afterwards.
Disappointingly we could not transform the strain. This was in accordance to the
results of
Petra
Wurmser, who
was also not able to reintroduce natural competence into *S. elongatus*
UTEX
2973 through
this
method.

In hope of better results we decided to try and revert the point mutation in the
*pilN*
gene with
a CRISPR/Cas12a system.While still working on the system itself as described in
the
 design section, we used
the pSL2680 plasmid  [(Ungerer and Pakrasi, 2016)](https://www.nature.com/articles/srep39681) to engineer our strain. We followed their protocol [(available here on
Addgene)](https://www.addgene.org/85581/) to construct a guiding
crRNA, leading the Cas12a enzyme to the mutated *pilN*  gene and cloned it together with the enzyme and a repair
template
to revert the
point
mutation. Sequencing results of all parts needed were correct, so the final
construct could be
transformed into our cyanobacteria.
In this approach triparental conjugation was performed slightly differently,
as
we wanted to
exactly
follow the provided protocol:
In contrary to the other method, a HB101 strain harboring pRL623 was again
transformed with
the
plasmid
of interest, in this case the fully assembled pSL2680 and the other HB101
strain
just
contained
pRL443.
Again, both strains were mixed together and then inoculated before being
blotted
on filters on
BG11
plates. This time, after only 6h the filters were transferred on BG11 plates
with kanamycin.
During our first run a few colonies were visible after just three days, but
getting cultures
to
grow
in
liquid media did not work as expected and the colonies did not look as
healthy
as hoped.
As we have had this kind of issue several times during our project and saw
that
in literature
the
colonies growing on the plates looked differently, we decided to reach out
to
experienced
researchers
working with the same *Synechococcus elongatus* strain. We contacted [Prof. Dr. James W. Golden](https://2019.igem.org/Team%3AMarburg/Human_Practices#james_golden)
from
the
University of California, San Diego to ask for advice and
were
able to
discover a crucial difference in the way we work with our strain: While in
our
own lab this is
not
commonly done, the researchers from the Golden lab put
Na2S2O3 (sodium thiosulfate) in their
BG11
plates
and we could find literature to support this advice [(Thiel
et al., 1989)](https://jb.asm.org/content/171/10/5743.short). This might seem like
a
small
addition, but it proved to be an essential factor, as after adding this
ingredient to our own
plates
we
were able to grow way better looking colonies, and not just that - the
colonies
started coming
up
after
just one day in our incubator.
The reason is simple: When autoclaving agar that contains phosphates,
reactive
oxygen species
such
as
H2O2 can be formed, which can drastically hinder
bacterial
growth
[(Tanaka et al., 2014)](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249246/). The
thiosulfate from Na2S2O3 is oxidized as
H2O2 is reduced to 2 H2O - so clearly
thiosulfate can
act as a reducing agent, eliminating reactive oxygen species from the
medium.
Presumably the
missing sodium thiosulfate in our BG11 agar was not the sole reason we did
not
get the desired
results on our plate, but in combination with the toxicity of Cas12a (though
less toxic than
Cas9, it still exhibits nuclease activity, cutting the DNA of the bacteria)
the
pressure was
too high for most of the cells. With our new found ingredient we were
certain to
get it right
this time and set the whole process in motion, this time with BG11 plates
including sodium
thiosulfate. In one huge experiment we tried a wide variety of different
conjugation
approaches, following the instructions of multiple papers ( [Ungerer and Pakrasi,
2016](https://www.nature.com/articles/srep39681); [Yu *et
al.*, 2015](https://www.nature.com/articles/srep08132) ; [Wendt *et al.*, 2016](https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7)). We are currently screening for correct
edits
and are certain to hold
a naturally competent strain in our hands in a matter of days.

![latest conjugation of UTEX2973 with the plasmid pSL2680.](https://static.igem.org/mediawiki/2019/f/ff/T--Marburg--Conjugation_UTEX2973_pSL2680.png)

Fig.4 : Latest conjugation of UTEX2973 with the plasmid pSL2680.

CRISPR gene editing

Although CRISPR/Cas systems have been discussed as incredibly powerful tools in
genetic
engineering,
they have
not yet been widely used in cyanobacterial research, which is why we set out to
implement such a
system, based
on CRISPR/Cas12a, into our [Green
Expansion](https://2019.igem.org/Team%3AMarburg/Part_Collection) of the Marburg Collection.

As CRISPR/Cas12a has already been reported to work in  *S.elongatus* UTEX 2973
 [(Ungerer and Pakrasi, 2016)](https://www.nature.com/articles/srep39681), we
were sure that it could be transformed into a Golden Gate Assembly compatible
version, allowing for more flexible [design](https://2019.igem.org/Team%3AMarburg/Design#strain_engineering) considerations.
While we started the cloning processes needed to change the existing vector into the
phytobrick
standard, we
tried the vector at hand ourselves, in order to assess its usefulness.
Following the given protocols we constructed a CRISPR/Cas12a vector harboring a
crRNA
and repair
template
designed to revert the point mutation in the  *pilN*  gene of our *S.elongatus* strain.
After a
few
initial problems we were able to get conjugants and are currently screening for
those
containing
the
desired
edit - more on this approach can be found in the natural competence section of our
results.

In order to modularize this system we built different parts for our genetic toolbox.
First of all
we
created
a
lvl0 part of the Cas12a protein by amplifying the sequence from the pSL2680 plasmid,
including
overhangs
that
enabled us to clone the PCR product into a lvl0 acceptor vector.
![map](https://static.igem.org/mediawiki/2019/a/ae/T--Marburg--Cas12a_CDS_lvl0.png)

Fig. 5: The plasmid map for the coding sequence of the lvl0 construct.

Sequencing results proved, that this crucial part was correctly assembled, ready
to
be used in lvl
1
constructs
- which we promptly did, using the following lvl0 parts:
pMC0\_1\_03 + pMC0\_2\_03 + pMC0\_3\_07 + pMC0\_4\_33 + pMC0\_5\_07 + pMC0\_6\_17.

![map](https://static.igem.org/mediawiki/2019/a/ae/T--Marburg--Cas12a_CDS_lvl0.png)

Fig. 6: Sequencing result of the coding sequence of the Cas12a protein.

The chosen promoter is a rather weak one, so that overproduction of Cas12a is
prevented, leading
to
less
toxicity in the cells.

![map](https://static.igem.org/mediawiki/2019/7/74/T--Marburg--Cas12a_CDS_lvl1.png)

Fig. 7: The lvl1 construct of the Cas12a protein was built with a weaker
promoter to reduce
toxic
effects.

Having built this construct, we continued to build the other missing part: the
crRNA.
The design of the pSL2680 plasmid was mostly kept the same, but in order to have
an
easy and cheap
selection
method we switched the lacZ cassette with a GFP cassette.

![crRNA lvl0](https://static.igem.org/mediawiki/2019/7/7e/T--Marburg--Cas12a_crRNA_lvl0.png)

Fig. 8: lvl0 design for the crRNA. The spacers can be exchanged with even
more
gRNAs to allow
for
multiplex genome editing.

We could show the correct assembly of this part - everything was as we planned
in
our [design](https://2019.igem.org/Team%3AMarburg/Design#strain_engineering)
meaning that we had all the parts in our MoClo standard.

![[Fig XX seq results of crRNA lvl0],](https://static.igem.org/mediawiki/2019/3/30/T--Marburg--Cas12a_crRNA_lvl0_seq.png)

Fig. 9: Sequencing result of the lvl0 construct of the crRNA

As the whole system is built for modular cloning in the PhytoBrick syntax, it is
possible to
freely
exchange
the
parts around the Cas12a and crRNA parts. This enables the use of different
promoters, allowing for
easy
screening: Constructs with weaker promoters in front of the Cas12a gene would
lead
to less gene
expression
and
therefore lower toxicity of the whole system. The free exchange of these
promoter
parts can
consequently be
used
for the creation of a library in order to look for the perfectly fitting
promoter
for this system.
Successfully creating these invaluable parts, we were able to establish a
workflow
for faster
cloning in *S.elongatus*.
As our system is modularized, it is possible to easily exchange the GFP cassette
for
the desired
crRNA,
which
can be done in a single reaction, further simplifying the cloning process of
CRISPR/Cas12a
constructs.
As shown [before](https://2019.igem.org/Team%3AMarburg/Design#strain_engineering) , the cloning process with the pSL2680 can take over a week, is
tedious work and is accompanied by another couple of days waiting for colonies.
In comparison, our system enables for efficient cloning in only four days: On
the first day the construct is assembled in a Golden Gate reaction, which is
thereafter transformed into *E.coli*. The next day
colonies can be picked,
inoculated and the construct can be
extracted in the evening. On the third day it can be transformed into *S.elongatus* -
and on the fourth day colonies can be screened.
The missing piece to apply an edit is the repair template. Skimming through
literature, we
noticed
that
transformation of linear DNA fragments into *S.elongatus* is supposedly
more efficient
than
the
transformation of whole plasmids  [(Almeida *et
al.*, 2017)](https://doi.org/10.1016/j.biori.2017.09.001) - and we were able to verify this fact in our own
experiments. This further simplifies our above mentioned workflow, as we are
able
to simply PCR the needed repair template from a DNA sequence and use the PCR
product for
transformation into *S.elongatus*. Our toolbox has a
special feature that can be used for exactly this workflow: a NotI cutting
site
can be found
in
our
constructs,
which is used to linearize them, so that they can be more efficiently
transformed.

We are more than certain that our modular CRISPR/Cas12a proves to be an
invaluable
contribution
to the
tools
available in cyanobacterial research, especially for the Golden Gate
community,
which is
growing
bigger
and
bigger every year - also thanks to the iGEM headquarters finally integrating
the
TypeIIS
standard into
the
competition!

Cyanobacterial shuttle vectors

As we have already clarified in the [description](https://2019.igem.org/Team%3AMarburg/Description#strain_engineering) part, self replicating shuttle vectors are
essential
for
many
workflows, as the gene expression levels are higher and non of the tedious
selection
processes that
come
with
genomic integrations have to be done.

On our road to the modular vector we were seeking, we firstly cured our own S.
elongatus UTEX 2973
strain of its
pANS plasmid. This was done by transforming the pAM4787 vector, which holds a
spectinomycin
resistance
as well as a
YFP cassette
[(Chen *et al.*,
2016)](https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377). Due to plasmid incompatibility - explained here in our [design](https://2019.igem.org/Team%3AMarburg/Design#strain_engineering) section - and because antibiotic pressure is
applied,
the pANS plasmid was over time
cured
from the
strain, which then just kept the pAM4787 plasmid. Transformation was done by
conjugation with the
pRK2013 plasmid in
DH5ɑ and the pAM4787 in HB101. Both were grown to an OD600≈0.5, washed in LB and
mixed with *S.
elongatus* which was
grown to late exponential phase and then washed in BG11.
We could clearly show, that the conjugant strain bears the pAM4787 plasmid if
selective pressure
is
held up.

![ https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png](https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png)

Fig. 10: Cell counts of conjugant strain. The y axis shows relative YFP
fluorescence and the x
axis relative autofluorescence.

This was followed by us starting to culture the pAM4787 bearing strain without
antibiotics again, slowly removing selective pressure from the cells. As the
plasmid
does not give
them any other
advantage and is probably just more metabolic burden due to the constantly
produced
YFP proteins it
is
slowly being
lost.
We could prove this in multiple setups: with the flow cytometry device we were
kindly granted access
to we could
clearly show the missing YFP signal in the cured *S. elongatus* strain and
logically this could also be observed over our UV table.

![https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png ](https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png)
![https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png](https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png)

Fig. 11
a) Cell counts of cured strain. The y axis shows relative YFP fluorescence
and
the x axis
relative
autofluorescence
b) Comparison of the fluorescence signal of the transformed (left) and cured
(right) strain.

Furthermore we performed colony PCRs as a test. We sent our plasmid-free strain
to
Next Generation
Sequencing in order to ensure that the strain really has lost the
pANS plasmid.

![gel pcr](https://static.igem.org/mediawiki/2019/e/e9/T--Marburg--ColonyPCRcuredStrain.jpg)

Fig. 12: Colony PCR of the wild type, the conjugated and the cured
strain.

Our next step was the characterization of the cyanobacterial shuttle vector
mentioned
in our  [design](https://2019.igem.org/Team%3AMarburg/Design#strain_engineering)  section. In an extensive flow cytometry
experiment we assessed the fluorescence of a transformed YFP-construct in our
cured
strain, showing
that the shuttle vector with the minimal replication element can be maintained
in
*S. elongatus* UTEX
2973.

After another four weeks of cultivation we looked at our
cultures again on the UV table to check if fluorescence was still present and
the
high intensity
of
fluorescence
proved to us, that the plasmid is still stably replicated in our strain, showing
us,
that the
minimal replication
element does indeed work in our strain.
For further analysis we performed qPCR with this transformed strain, in order to
check the copy
number of the
vector. We used the copy number of pANL as a reference, which is supposedly at
~2,6
copies per
chromosome
[(Chen *et al.*,
2016)](https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377). Our data shows a ~4,5 times higher copy number relative to pANL,
meaning that the
construct is maintained with approximately 11,7 copies per chromosome.

For further analysis of this part ([BBa\_K3228069](http://parts.igem.org/Part%3ABBa_K3228069)) we performed a
Quantitative-Polymerase-Chain-Reaction (qPCR) with this transformed strain, in
order
to check the copy number of the vector. This proved to be difficult in
Cyanobacteria, due to variation in genome copy number ([Griese *et al.*, 2011](https://onlinelibrary.wiley.com/doi/full/10.1111/j.1574-6968.2011.02368.x?sid=nlm%3Apubmed); [Chen *et al.*, 2012](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427279/)).
To overcome this problem, we noticed that the copynumber of pANL stays rather
stable
with 2,6 copies per chromosome ([Chen *et al.*, 2016](https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377)).
Therefore we performed the qPCR with pANL as an additional target. For each
target,
we chose three different primer pairs, with known efficiency. Five technical and
two
biological replicates were used. The samples were normalised to the chromosome
and
pANL was used to identify the total copy number per chromosome (figure 14). Our
data
shows a ~4,5 times higher copy number relative to pANL, meaning that the
construct
is maintained with approximately 11,7 copies per chromosome. This is comparable
with
the copynumber of pANS in  [Chen *et al.* 2016](https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377).

![Copynumber Evaluation of Ori-part via qPCR](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--Parts--qPCR-Lvl1.png)

Fig. 14: Copynumber Evaluation of Ori-part via qPCR

Additionally we measured the fluorescence signals in a plate reader at different
optical
densities
and could again
confirm high fluorescence signals, indicating strong gene expression in
constructs
built
around
this replication
element.

![diagramm](https://static.igem.org/mediawiki/2019/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg)

Fig. 12:YFP fluorescence at different optical densities.

All this data confirms that the construct actually works and can be reliably used
as
a
cyanobacterial shuttle
vector, proving that BBa\_K3228069 works as intended, thus functioning as our  [validated part](parts.igem.org/Part%3ABBa_K3228069).
This assumption is solidified by all our sequence data, showing that the shuttle
vectors were
completely assembled
as planned in our design section.

# M A R B U R G C O L L E C T I O N  2.0

---

We expanded the Marburg Collection by adding the Green expansion and the first MoClo
compatible
shuttle
vector for Cyanobacteria.

# Marburg Collection 2.0

X

Overview over the expansion of the Marburg Collection:

We added 55 new parts to the Marburg Collection, adding several new features such as
the
Green
expansion,
including a
kit for the Modularized Engineering of Genome Areas (M.E.G.A.) and the first MoClo
compatible shuttle
vector for cyanobacteria.
Additionally, we offer a set of reporters suitable for characterization of BioBricks
in
cyanobacteria
and
ribozymes for a more
stable and species independent transcription. We also provide standardized
measurement
vectors that
were
generated using our
designed placeholders.

![Overview over the expansion of the Marburg Collection](https://static.igem.org/mediawiki/2019/2/2b/T--Marburg--Toolbox_table.svg)

Fig.1: Overview over the expansion of the Marburg Collection

Overview over the different expansions in the Marburg Collection 2.0

To give a better overview we show here the different expansions we added to the
Marburg
Collection:

# Parts

| ![5'Homology](https://static.igem.org/mediawiki/2019/5/5b/T--Marburg--5%27Homology.svg) | ![Termiantor](https://static.igem.org/mediawiki/2019/3/3a/T--Marburg--Terminator.svg) | ![AntibioticResistance](https://static.igem.org/mediawiki/2019/1/1e/T--Marburg--Antibiotic_Resistance.svg) | ![3'Homology](https://static.igem.org/mediawiki/2019/3/30/T--Marburg--3%27Homology.svg) | ![ShuttleVector](https://static.igem.org/mediawiki/2019/f/fc/T--Marburg--Shuttle_Vector.svg) ![CDS](https://static.igem.org/mediawiki/2019/5/51/T--Marburg--CDS.svg) | |
| --- | --- | --- | --- | --- | --- |
| * [K3228000](http://parts.igem.org/Part%3ABBa_K3228000)   (aNSo1   integration up) * [K3228001](http://parts.igem.org/Part%3ABBa_K3228001)   (aNSo2   integration up) * [K3228020](http://parts.igem.org/Part%3ABBa_K3228020)   (NS1 up) * [K3228021](http://parts.igem.org/Part%3ABBa_K3228021)   (NS2 up) * [K3228022](http://parts.igem.org/Part%3ABBa_K3228022)   (NS3 up) | * [K3228004](http://parts.igem.org/Part%3ABBa_K3228004)   (shortTB0010) * [K3228005](http://parts.igem.org/Part%3ABBa_K3228005)   (shortTB0015) * [K3228006](http://parts.igem.org/Part%3ABBa_K3228006)   (shortTB1002) * [K3228007](http://parts.igem.org/Part%3ABBa_K3228007)   (shortTB1003) * [K3228008](http://parts.igem.org/Part%3ABBa_K3228008)   (shortTB1004) * [K3228009](http://parts.igem.org/Part%3ABBa_K3228009)   (shortTB1005) * [K3228010](http://parts.igem.org/Part%3ABBa_K3228010)   (shortTB1006) * [K3228011](http://parts.igem.org/Part%3ABBa_K3228011)   (shortTB1007) * [K3228012](http://parts.igem.org/Part%3ABBa_K3228012)   (shortTB1008) * [K3228013](http://parts.igem.org/Part%3ABBa_K3228013)   (shortTB1009) * [K3228014](http://parts.igem.org/Part%3ABBa_K3228014)   (shortTB1010) * [K3228015](http://parts.igem.org/Part%3ABBa_K3228015)   (shortDummy) | * [K3228016](http://parts.igem.org/Part%3ABBa_K3228016)   (SpecRes\_short) * [K3228017](http://parts.igem.org/Part%3ABBa_K3228017)   (CmlRes\_short) * [K3228018](http://parts.igem.org/Part%3ABBa_K3228018)   (TetRes\_short) * [K3228019](http://parts.igem.org/Part%3ABBa_K3228019)   (KanRes\_short) * [K3228027](http://parts.igem.org/Part%3ABBa_K3228027)   (GenRes\_short) | * [K3228002](http://parts.igem.org/Part%3ABBa_K3228002)   (aNSo1 integration down) * [K3228003](http://parts.igem.org/Part%3ABBa_K3228003)   (aNSo2 integration down) * [K3228023](http://parts.igem.org/Part%3ABBa_K3228023)   (NS1 down) * [K3228024](http://parts.igem.org/Part%3ABBa_K3228024)   (NS2 down) * [K3228025](http://parts.igem.org/Part%3ABBa_K3228025)   (NS3 down) | * [K3228026](http://parts.igem.org/Part%3ABBa_K3228026)   (oriT integration) * [K3228069](http://parts.igem.org/Part%3ABBa_K3228069)   (panS SpecRes LVL1) * [K3228089](http://parts.igem.org/Part%3ABBa_K3228089)   (panS KanRes LVL2) | * [K3228051](http://parts.igem.org/Part%3ABBa_K3228051)   (Limonene   Synthase) * [K3228052](http://parts.igem.org/Part%3ABBa_K3228052)   (Farnesen   Synthase) |

| ![RiboInsulator](https://static.igem.org/mediawiki/2019/2/28/T--Marburg--Ribo_Insulator.svg) | ![Spaceholder](https://static.igem.org/mediawiki/2019/1/15/T--Marburg--Spaceholder.svg) | ![Measurement](https://static.igem.org/mediawiki/2019/4/4e/T--Marburg--Measurement_Vector.svg) | ![CRISPR](https://static.igem.org/mediawiki/2019/3/3c/T--Marburg--CRISPR.svg) | ![LuminescenceReporter](https://static.igem.org/mediawiki/2019/4/4e/T--Marburg--Luminescence_Reporter.svg) | ![FluorescenceReporter](https://static.igem.org/mediawiki/2019/1/15/T--Marburg--Fluorescence_Reporter.svg) |
| --- | --- | --- | --- | --- | --- |
| * [K3228054](http://parts.igem.org/Part%3ABBa_K3228054)   (riboJ B0034) * [K3228055](http://parts.igem.org/Part%3ABBa_K3228055)   (riboJ B0034 noScar) * [K3228056](http://parts.igem.org/Part%3ABBa_K3228056)   (SarJ B0034) * [K3228057](http://parts.igem.org/Part%3ABBa_K3228057)   (PlmJ B0034) | * [K3228060](http://parts.igem.org/Part%3ABBa_K3228060)   (PRO placeholder) * [K3228061](http://parts.igem.org/Part%3ABBa_K3228061)   (RBS placeholder) * [K3228062](http://parts.igem.org/Part%3ABBa_K3228062)   (CDS placeholder) * [K3228063](http://parts.igem.org/Part%3ABBa_K3228063)   (TER placeholder) | * [K3228073](http://parts.igem.org/Part%3ABBa_K3228073)   (Promotor entry vector) * [K3228074](http://parts.igem.org/Part%3ABBa_K3228074)   (BS entry vector) * [K3228075](http://parts.igem.org/Part%3ABBa_K3228075)   (CDS entry vector) * [K3228090](http://parts.igem.org/Part%3ABBa_K3228090)   (Terminator entry vector) | * [K3228100](http://parts.igem.org/Part%3ABBa_K3228100)   (cpf1) * [K3228101](http://parts.igem.org/Part%3ABBa_K3228101)   (crRNA-GFP dropout) * [K3228103](http://parts.igem.org/Part%3ABBa_K3228103)   (cpf1+crRNA-GFP dropout) | * [K3228040](http://parts.igem.org/Part%3ABBa_K3228040)   (NanoLuc) * [K3228041](http://parts.igem.org/Part%3ABBa_K3228041)   (NanoLuc S.e.) * [K3228042](http://parts.igem.org/Part%3ABBa_K3228042)   (TeLuc (S.e.)) * [K3228043](http://parts.igem.org/Part%3ABBa_K3228043)   (Antares S.e.) | * [K3228044](http://parts.igem.org/Part%3ABBa_K3228044)   (eYFP S.e.) * [K3228045](http://parts.igem.org/Part%3ABBa_K3228045)   (sYFP2 S.e.) * [K3228046](http://parts.igem.org/Part%3ABBa_K3228046)   (phluorin2 S.e.) * [K3228047](http://parts.igem.org/Part%3ABBa_K3228047)   (rxYFP S.e.) * [K3228048](http://parts.igem.org/Part%3ABBa_K3228048)   (sfGFP S.e.) * [K3228049](http://parts.igem.org/Part%3ABBa_K3228049)   (mTurquoise S.e.) |

Sequencing results of the lvl0 parts

We built and validated 55 new BioBricks this year. They are all listed in the
Registry of
Standard
Biological Parts
(Part range BBa\_3228000 to BBa\_32280103). All lvl0 Parts were validated by complete
sequencing.

![aNSo-lvl-0-front](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--anso-lvl_0_front.jpg)

Fig.7: aNSo-lvl-0-front

![aNSo-lvl-0-end](https://static.igem.org/mediawiki/2019/a/ab/T--Marburg--anso_lvl_0_end.jpg)

Fig.8: aNSo-lvl-0-end

Building constructs to test the lethality of origin of transfer

If plasmids reach a certain size normal transformation protocols are not feasible
anymore
to bring
the
plasmid into
the host.
For the transformation of such huge megaplasmids we designed an “origin of transfer”
BioBrick that
makes
it possible
to directly transport plasmids of any size from one species to another. To test if
this
sequence would
result in any
toxicity in a genomic context (source things where genome parts can be exchanged by
integrating such
sequences) we built
it into an integration vector.

Sequencing results of the lvl1 parts for modularized genome integrations

We successfully build two integration cassettes from our rationally designed
artificial
neutral
integration sites
(a.N.S.o. 1 and 2) and verified them by sequencing. These parts contained the
“origin of
transfer” to
test
their lethality
in the aforementioned experiment.

![aNSo1-lvl-1 with spec](https://static.igem.org/mediawiki/2019/2/2e/T--Marburg--anso_1_mit_Spec_LVL_1.jpg)

Fig.9: aNSo1-lvl-1 with spec

![anso2-lvl-1 with cml](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--anso_2_mit_cml_LVL_1.jpg)

Fig.10: anso2-lvl-1 with cml

Workflow to integrate a modularized integration cassette

We established a workflow on how to integrate a cassette - from lvl0 Parts to a
finished
change in
genome. With
UTEX 2973 this is possible in less than five days, while in PCC7942 the same
integration
would take a
whole
month.

![anso wirkflow](https://static.igem.org/mediawiki/2019/4/41/T--Marburg--Toolbox_Model_ANSOscreening.svg)

Fig.11: Workflow for finding new neutral integration sites.

Using the placeholder to build standard measurement vectors

We successfully used our placeholders to build and validate the standardized
measurement
vectors for
promoters,
ribosomal binding sites and coding sequences. We evaluated the cost and time savings
from a library
assembly with a
sample size of 25.
Through our design decision to build placeholders we managed to cut the workload for
a
high throughput
assembly by around 72%
and the invested financial resources by 40 % with just a sample size of 25
assemblies.

[ ]
 Workload and cost for
placeholder

# Workload for placeholder

| Task | Time per sample [min] | Samples | Total |
| --- | --- | --- | --- |
| Assembly of entry vector (7 part assembly) |  | 1 |  |
| Golden Gate reaction | 20 | 1 | 20 |
| Transformation | 15 | 1 | 15 |
| Preparing 10 overnight cultures + purification | 15 | 10 | 150 |
| Test digesting | 5 | 5 | 25 |
|  |  |  |  |
| Inserting Promoters (2 part assembly) (25 BioBricks) |  | 25 |  |
| Golden Gate reactions (simplified with a mastermix) | 5 | 25 | 125 |
| Transformation | 10 | 25 | 250 |
| Preparing overnight cultures + purification | 15 | 50 | 750 |
|  |  |  |  |
| Total |  |  | **1335** |
| In hours |  |  | **22,25** |
|  |  |  |  |
| **Workflow without placeholders** |  |  |  |
|  |  |  |  |
| lvl1 Golden Gate Assembly |  | 25 |  |
| Golden Gate reaction | 5 | 25 | 125 |
| Transformation | 10 | 25 | 250 |
| Preparing 10 overnight cultures + purification | 15 | 250 | 3750 |
| Test digesting | 5 | 125 | 625 |
|  |  |  |  |
| Total |  |  | **4750** |
| In hours |  |  | **79,16666667** |
|  |  |  |  |
|  |  |  |  |
| Differences in hours |  |  | **56,91666667** |
| Percentage saved |  |  | **0,72** |

# Cost for placeholder

| Major cost point | Price [€] | Samples | Total |
| --- | --- | --- | --- |
| With placeholder |  |  |  |
| **lvl1 Golden Gate mix for measurement vector assembly** |  | 1 |  |
| BsaI | 2 | 1 | 2 |
| BsmbI | 2 | 1 | 2 |
|  |  |  |  |
| **Plasmid purification** | 2 | 5 | 10 |
|  |  |  |  |
| **Test digesting** |  | 5 |  |
| Enzyme | 1 | 5 | 5 |
| Aggarose gel and ladder (approximation) | 0,5 | 2,5 | 1,25 |
|  |  |  |  |
| Sequencing (estimated) | 3 | 3 | 9 |
|  |  |  |  |
|  |  |  |  |
| **lvl1 Golden Gate mix for inserting the promoter**> |  | 25 |  |
| BsaI | 2 | 25 | 50 |
| BsmbI | 2 | 25 | 50 |
|  |  |  |  |
|  |  |  |  |
| **Plasmid purification** | 2 | 50 | 100 |
| Sequencing | 3 | 50 | 150 |
|  |  |  |  |
| **Total** |  |  | **378,25** |
|  |  |  |  |
|  |  |  |  |
| **Without placeholder** |  |  |  |
| **lvl1 Golden Gate mix for measurement vector assembly** |  | 25 |  |
| BsaI | 2 | 25 | 50 |
| BsmbI | 2 | 25 | 50 |
|  |  |  |  |
| **Plasmid purification** | 2 | 125 | 250 |
|  |  |  |  |
| **Test digesting** |  | 125 |  |
| Enzyme | 1 | 125 | 125 |
| Aggarose gel and ladder (approximation) | 0,5 | 62,5 | 31,25 |
| Sequencing (estimated) | 3 | 41,625 | 124,875 |
|  |  |  |  |
|  |  |  |  |
| **Total** |  |  | **378,25** |
|  |  |  |  |
| **Difference** |  |  | **251,875** |
| **Percentage saved** |  |  | **0,4** |

Construction of a promoter library with standard measuring vectors

We built a promoter library using our standard promoter measurement vector and 25
BioBricks.
Here we show a list of all the BioBricks we used.

![Promoter library 1r](https://static.igem.org/mediawiki/2019/3/31/T--Marburg--promoter_library_1.jpg)

Fig.14: Promoter library 1

![Promoter library 2](https://static.igem.org/mediawiki/2019/1/1a/T--Marburg--promoter_library_2.jpg)

Fig.15: Promoter library 2

Workflow for the screen of a BioBrick library

We designed a workflow to build a library, introduce it into UTEX2973 and measure its
characteristics.

![Measurement-workflow](https://static.igem.org/mediawiki/2019/e/e3/T--Marburg--Toolbox_MeasurementWorkflow.svg)

Fig.16: Measurement-workflow

Testing the reproducibility and standard deviation of the screening workflow

We tested how reproducible results from our library screening workflow are with a
fluorescence
reporter.

![YFP Pam 4787 6 replicates](https://static.igem.org/mediawiki/2019/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg)

Fig.17: YFP Pam 4787 6 replicates

Application note for the characterization of BioBricks in our chassis

After calibrating our screening procedure, we decided to share our practical
knowledge
with other end
users.
[Application
Note](https://static.igem.org/mediawiki/2019/0/07/T--Marburg--Berthold_Application_Note.pdf)

![THM](https://static.igem.org/mediawiki/2019/f/f5/T--Marburg--logo_thm.svg)

![Marburg](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--logo_marburg.svg)

![Gießen](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--logo_jlu.svg)

![Mail](https://static.igem.org/mediawiki/2019/2/2c/T--Marburg--m_icon_ma.svg)
[![Instagram](https://static.igem.org/mediawiki/2019/e/e7/T--Marburg--m_icon_in.svg)](https://www.instagram.com/igem.marburg.2019)
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