iGEM 2018 — Team Goettingen

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# Team:Goettingen/Notebook

⇧

Team Göttingen
 iGEM 2018

![](https://static.igem.org/mediawiki/2018/d/d4/T--Goettingen--maskotchen.png)

Glyphosate on my plate?

![](https://static.igem.org/mediawiki/2018/4/44/T--Goettingen--Main-Page-iGEM-Logo_tinted.png)

[Home](https://2018.igem.org/Team%3AGoettingen)
[News](https://2018.igem.org/Team%3AGoettingen/News)
Team

[Team Members](https://2018.igem.org/Team%3AGoettingen/Team)
[Collaborations](https://2018.igem.org/Team%3AGoettingen/Collaborations)

Project

[Background](https://2018.igem.org/Team%3AGoettingen/Background)
[Description](https://2018.igem.org/Team%3AGoettingen/Description)
[Design](https://2018.igem.org/Team%3AGoettingen/Design)
[Experiments](https://2018.igem.org/Team%3AGoettingen/Experiments)
[Notebook](https://2018.igem.org/Team%3AGoettingen/Notebook)
[InterLab](https://2018.igem.org/Team%3AGoettingen/InterLab)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)
[Results](https://2018.igem.org/Team%3AGoettingen/Results)
[Demonstrate](https://2018.igem.org/Team%3AGoettingen/Demonstrate)
[Improve](https://2018.igem.org/Team%3AGoettingen/Improve)
[Attributions](https://2018.igem.org/Team%3AGoettingen/Attributions)

Parts

[Parts Overview](https://2018.igem.org/Team%3AGoettingen/Parts)
[Basic Parts](https://2018.igem.org/Team%3AGoettingen/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AGoettingen/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AGoettingen/Part_Collection)

[Safety](https://2018.igem.org/Team%3AGoettingen/Safety)
Human Practices

[Human Practices](https://2018.igem.org/Team%3AGoettingen/Human_Practices)
[Survey](https://2018.igem.org/Team%3AGoettingen/Human_Practices/Survey)
[Education & Engagement](https://2018.igem.org/Team%3AGoettingen/Public_Engagement)

Awards

[Applied Design](https://2018.igem.org/Team%3AGoettingen/Applied_Design)
[Hardware](https://2018.igem.org/Team%3AGoettingen/Hardware)
[Measurement](https://2018.igem.org/Team%3AGoettingen/Measurement)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)

[Judging Form](https://igem.org/2018_Judging_Form?id=2586)

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≡

Contact:

|  |  |
| --- | --- |
| E-mail: | igem2018@gwdg.de |
| Adress: | c/o iGEM 2018 |
|  | Department of General Microbiology |
|  | Grisebachstraße 8 |
|  | 37077 Göttingen |
|  | Germany |

We are especially grateful to our numerous sponsors on [gofundme,](https://de.gofundme.com/glyphosate-on-my-plate) who supported our project and made it possible.
 Every public donor, who helped us to achieve our goals got a special place on our bacterial wall of fame and a handdrawn microbe to show our appreciation. A link to that wall can be found here:

[Bacterial Wall of Fame](https://2018.igem.org/Team%3AGoettingen/WallOfFame)

![Sartorius-Sponsor](https://static.igem.org/mediawiki/2018/d/db/T--Goettingen--Main-Page-sartorius.png)
![Uni-Goettingen-Sponsor](https://static.igem.org/mediawiki/2018/f/f6/T--Goettingen--Main-Page-goettingen.png)
![GZMB-Sponsor](https://static.igem.org/mediawiki/2018/8/8c/T--Goettingen--Main-Page-gzmb.png)

## Our Labnotes:

In this section, you can see our lab notes. Our topic is divided into four subtopics which are the following ones. You can choose between "Fluorophores", "Biobricks", "Evolution", and "Reporter-Systems" and the respective months. Beyond this section, our strain list is shown.

Project:

Fluorophores
Biobricks
Evolution
Reporter-Systems

Month:

April
May
June
July
August
September
October

Submit

---

**STRAIN LIST**

| Name | Name after isolation | Genotype |
| --- | --- | --- |
| 168 |  | *trpC2* |
| SP1 |  | wildtype |
| iGEM1 | Suppressor 1 10 mM | *trpC2* 1.3 kbp deletion *5'-yhfE-yhfF-3'-gltT* |
| iGEM2 | Suppressor 2 10 mM | *trpC2* not determined |
| iGEM3 | Suppressor 3 10 mM | *trpC2* not determined |
| iGEM4 | Suppressor 4 10 mM | *trpC2* not determined |
| iGEM5 | Suppressor 5 10 mM | *trpC2* not determined |
| iGEM6 | Suppressor 1-1 30 mM | *trpC2* not determined |
| iGEM7 | Suppressor 1-2 30 mM | *trpC2* 1.3 kbp deletion *5'-yhfE-yhfF-3'-gltT gltP*(G232A) |
| iGEM8 | Suppressor 2-1 30 mM | *trpC2* not determined |
| iGEM9 | Suppressor 2-2 30 mM | *trpC2* not determined |
| iGEM10 | Suppressor 3-1 30 mM | *trpC2* not determined |
| iGEM11 | Suppressor 3-2 30 mM | *trpC2* not determined |
| iGEM12 | Suppressor 1-1 40 mM | *trpC2* not determined |
| iGEM13 | Suppressor 1-2 40 mM | *trpC2* 1.3 kbp deletion *5'-yhfE-yhfF-3'-gltT* 11 bp deletion in *gltP* |
| iGEM14 | Suppressor 2-1 40 mM | *trpC2* not determined |
| iGEM15 | Suppressor 2-2 40 mM | *trpC2* not determined |
| iGEM16 | Suppressor 3-1 40 mM | *trpC2* not determined |
| iGEM17 | Suppressor 3-2 40 mM | *trpC2* not determined |
| BP233 |  | *trpC2* Δ*gltT::spc* |
| BP234 |  | *trpC2* Δ*gltP::cat* |
| BP235 |  | *trpC2* Δ*gltT::spc* Δ*gltP::cat* |
| BP236 |  | *trpC2 amyE::(Palf4-gltT-lacZ aphA3)* |
| BP237 |  | *trpC2* Δ*gltT::spc amyE::(Palf4-gltT-lacZ aphA3)* |
| iGEM18 |  | *amyE::(PtrpE-lacZ aphA3)* |
| iGEM19 |  | *trpC2 amyE::(PtrpE-lacZ aphA3)* |
| iGEM20 | SP1\_GP342 | *amyE::(PgltA-lacZ aphA3)* |
| iGEM21 | SP1\_GP650 | *amyE::(PgltA-lacZ aphA3) gltC::TnIO spc* |
| iGEM22 | 168::BFP | *trpC2 amyE::(BFP-lacZ aphA3)* |
| iGEM23 | 168::GFP | *trpC2 amyE::(GFP-lacZ aphA3)* |
| iGEM24 | 168::mOrange | *trpC2 amyE::(mOrange-lacZ aphA3)* Δ*gltT::spc* |
| iGEM25 | 168::mCherry | *trpC2 amyE::(mCherry-lacZ aphA3)* Δ*gltT::spc* |
| iGEM26 | BP233::BFP | *trpC2 amyE::(BFP-lacZ aphA3)* Δ*gltT::spc* |
| iGEM27 | BP233::GFP | *trpC2 amyE::(GFP-lacZ aphA3)* Δ*gltT::spc* |
| iGEM28 | BP233::mOrange | *trpC2 amyE::(mOrange-lacZ aphA3)* Δ*gltT::spc* |
| iGEM29 | BP233::mCherry | *trpC2 amyE::(mCherry-lacZ aphA3)* Δ*gltT::spc* |
| iGEM30 | Spden::BFP | *trpC2 amyE::(BFP-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM31 | Spden::GFP | *trpC2 amyE::(GFP-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM32 | Spden::mOrange | *trpC2 amyE::(mOrange-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM33 | Spden::mCherry | *trpC2 amyE::(mCherry-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM34 | BP235::BFP | *trpC2 amyE::(BFP-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM35 | BP235::GFP | *trpC2 amyE::(GFP-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM36 | BP235::mOrange | *trpC2 amyE::(mOrange-lacZ aphA3)* Δ*gltT::spc* Δ*gltP::cat* |
| iGEM37 | SP1::Gat | *amyE::(gat-lacZ aphA3)* |
| iGEM38 | SP1::AroA\* | *amyE::(aroA\*-lacZ aphA3)* |
| iGEM39 | SP1::Gat::Δ*gltT* | *amyE::(gat-lacZ aphA3)* Δ*gltT::spc* |
| iGEM40 | SP1::AroA\*::Δ*gltT* | *amyE::(aroA\*-lacZ aphA3)* Δ*gltT::spc* |
| iGEM41 | SP1::Δ*gltT* | Δ*gltT::spc* |


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Goettingen/Experiments

⇧

Team Göttingen
 iGEM 2018

![](https://static.igem.org/mediawiki/2018/d/d4/T--Goettingen--maskotchen.png)

Glyphosate on my plate?

![](https://static.igem.org/mediawiki/2018/4/44/T--Goettingen--Main-Page-iGEM-Logo_tinted.png)

[Home](https://2018.igem.org/Team%3AGoettingen)
[News](https://2018.igem.org/Team%3AGoettingen/News)
Team

[Team Members](https://2018.igem.org/Team%3AGoettingen/Team)
[Collaborations](https://2018.igem.org/Team%3AGoettingen/Collaborations)

Project

[Background](https://2018.igem.org/Team%3AGoettingen/Background)
[Description](https://2018.igem.org/Team%3AGoettingen/Description)
[Design](https://2018.igem.org/Team%3AGoettingen/Design)
[Experiments](https://2018.igem.org/Team%3AGoettingen/Experiments)
[Notebook](https://2018.igem.org/Team%3AGoettingen/Notebook)
[InterLab](https://2018.igem.org/Team%3AGoettingen/InterLab)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)
[Results](https://2018.igem.org/Team%3AGoettingen/Results)
[Demonstrate](https://2018.igem.org/Team%3AGoettingen/Demonstrate)
[Improve](https://2018.igem.org/Team%3AGoettingen/Improve)
[Attributions](https://2018.igem.org/Team%3AGoettingen/Attributions)

Parts

[Parts Overview](https://2018.igem.org/Team%3AGoettingen/Parts)
[Basic Parts](https://2018.igem.org/Team%3AGoettingen/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AGoettingen/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AGoettingen/Part_Collection)

[Safety](https://2018.igem.org/Team%3AGoettingen/Safety)
Human Practices

[Human Practices](https://2018.igem.org/Team%3AGoettingen/Human_Practices)
[Survey](https://2018.igem.org/Team%3AGoettingen/Human_Practices/Survey)
[Education & Engagement](https://2018.igem.org/Team%3AGoettingen/Public_Engagement)

Awards

[Applied Design](https://2018.igem.org/Team%3AGoettingen/Applied_Design)
[Hardware](https://2018.igem.org/Team%3AGoettingen/Hardware)
[Measurement](https://2018.igem.org/Team%3AGoettingen/Measurement)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)

[Judging Form](https://igem.org/2018_Judging_Form?id=2586)

[![Facebook-Button](https://static.igem.org/mediawiki/2018/9/90/T--Goettingen--social-facebook.png)](https://www.facebook.com/IGEM2018/)
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[![Youtube-Button](https://static.igem.org/mediawiki/2018/7/74/T--Goettingen--social-youtube.png)](https://www.youtube.com/channel/UCDowXCPO-HoC-oaiCaerT_Q)

≡

Contact:

|  |  |
| --- | --- |
| E-mail: | igem2018@gwdg.de |
| Adress: | c/o iGEM 2018 |
|  | Department of General Microbiology |
|  | Grisebachstraße 8 |
|  | 37077 Göttingen |
|  | Germany |

We are especially grateful to our numerous sponsors on [gofundme,](https://de.gofundme.com/glyphosate-on-my-plate) who supported our project and made it possible.
 Every public donor, who helped us to achieve our goals got a special place on our bacterial wall of fame and a handdrawn microbe to show our appreciation. A link to that wall can be found here:

[Bacterial Wall of Fame](https://2018.igem.org/Team%3AGoettingen/WallOfFame)

![Sartorius-Sponsor](https://static.igem.org/mediawiki/2018/d/db/T--Goettingen--Main-Page-sartorius.png)
![Uni-Goettingen-Sponsor](https://static.igem.org/mediawiki/2018/f/f6/T--Goettingen--Main-Page-goettingen.png)
![GZMB-Sponsor](https://static.igem.org/mediawiki/2018/8/8c/T--Goettingen--Main-Page-gzmb.png)

# Experiments

![](https://static.igem.org/mediawiki/2018/9/9c/T--Goettingen--MethodColl.png)

All experiments were performed using the Method Collection "Methods in Molecular Biology of Bacteria" provided by the [Department of General Microbiology](http://www.uni-goettingen.de/en/577601.html), University of Göttingen. The method collection is based on methods that have been collected and optimized during the past years by the students and supervisors of the Department of General Microbiology. The methods are suitable to engineer bacteria like *Bacillus subtilis*, *Escherichia coli*, *Listeria monocytogenes* and *Mycoplasma pneumoniae*. The book also contains protocols for the purification and characterization of proteins.
If you are interested in purchasing the whole method collection you can contact [us!](http://www.uni-goettingen.de/en/contact/579943.html) Hopefully, it will be soon available at [Amazon](https://www.amazon.com/).

## High-throughput cultivation of bacteria

For growth experiments with the Synergy MX II multi-well plate reader (Biotek) prepare pre- cultures and grow them over night at 28°C. Next day dilute these cultures (for instance 1:100, 1:500 and 1:1,000) in 1 - 5 ml of the same medium that will be later on used for recording growth curves and incubate them at 37°C (or other temperatures) and 220 rpm. At an OD600 of about 0.2 – 0.5 use the cultures to inoculate a 96 well microtitre plate that has been supplemented with 100 µl medium per well. The outermost wells of the microtitre plate should contain 150 µl of distilled water to avoid that medium containing cells evaporate. Incubate the plate for a maximum of 48 h at the intermediate shaking mode. The OD600 can be detected every 10 to 15 min. It is recommended to do at least three technical replicates per strain and condition on the same plate. Sometimes the detection of the OD can be disturbed by condensing water under the lid and your growth curve will look really strange.

## Genetic modification of bacteria

### Preparation of competent *E. coli* cells

#### Method 1 (low amount of competent cells, RbCl)

##### The Material

* LB liquid medium
* RF1 and RF2 buffer
* Liquid nitrogen

##### The Procedure

1. Inoculate a 4 ml culture either with a single colony or with a cryo culture of the desired *E. coli*  strain and incubate the culture with agitation over night at 37°C.
2. Inoculate a 300 ml shake flask containing 100 ml LB medium with the overnight culture to an OD600 of 0.05 and grow the culture at 37°C until the OD600 is about 0.3.
3. Transfer the cells into two 50 ml Falcon tubes, incubate the cultures for 15 min on ice and harvest the cells by centrifugation for 15 min at 5,000 rpm and 4°C. Discard the supernatants.
4. Re-suspend the cells in 1/3 of the original volume (~16 ml/50 ml) of buffer RF1, incubate the cells again on ice and harvest the cells by centrifugation for 15 min at 5,000 rpm and 4°C. Discard the supernatants.
5. Put 0.4 ml of the cell suspension into the Eppendorf reaction tubes and freeze the cells by transferring them immediately to the liquid nitrogen. Store the competent cells at -80°C.

#### Method 2 (fast method, cells cannot be stored, CaCl2)

##### The Material

* LB liquid medium 50 mM CaCl2 solution

##### The Procedure

1. Inoculate a 4 ml culture either with a single colony or with a cryo culture of the desired *E. coli*  strain and incubate the culture with agitation over night at 37°C.
2. Inoculate a 100 ml shake flask containing 10 ml LB medium with the overnight culture to an OD600 of 0.05 - 0.1 and grow the culture at 37°C until the OD600 is about 0.3.

   ![  ]( https://static.igem.org/mediawiki/2018/5/56/T--Goettingen--competentcells.png )

   Schematic illustration of competent cell preparation. The desired  *E. coli*  strain is grown to log phase, pelleted and re-suspended in CaCl2 solution. Now the cells are competent and should be kept on ice all the time. To test the competence of the cells, one aliquot is used to test the transformation efficiency. This is done with a standard plasmid, e.g. pUC19 and a defined amount. Afterwards the efficiency can be calculated.
3. Transfer the cells into 15 ml Falcon tubes and harvest the cells by centrifugation for 6 min at 5,000 rpm and 4°C. Discard the supernatants and re-suspend the cells in 5 ml of ice-cold CaCl2 solution.
4. Incubate the cells for 30 min on ice and collect them again by centrifugation for 6 min at 5,000 rpm and 4°C. Re-suspend the pellet in 1 ml of the ice-cold CaCl2 solution. Now the cells are ready to be transformed with your re-ligation and ligation samples.

#### Method 3 (high amount of competent cells, time-consuming)

##### The Material

* TB buffer (ice-cold)
* SOB -Mg
* 1 M MgCl2
* 1 M MgSO4
* DMSO (100 %)

##### The Procedure

1. Inoculate a 20 ml culture either with a single colony or with a cryo culture of the desired *E. coli* strain and incubate the culture with agitation for 20 h at 28°C.
2. Inoculate\* 250 ml SOB medium supplemented with 10 mM MgCl2 and 10 mM MgSO4 in a 2 l shake flask and grow the cells to an OD600 of 0.5 – 0.9 (20 – 24 h) at 18°C and 200 – 250 rpm.

   \*) The volume of the pre-culture strongly depends on the  *E. coli*  strain. Using DH5a it is recommended to use 4 ml pre-culture and inoculate at 8 a.m. Next day around 10 a.m. this strain should have reached an OD600 between 0.5 and 0.6. If you use the strain XL1-Blue, you will need less cells and it is sufficient to inoculate the SOB medium around lunchtime.
3. Incubate the whole flask for 10 min on ice. Collect the cells by centrifugation for 10 min at 4°C and 5,000 rpm. Re-suspend the cells in 80 ml of ice-cold TB and incubate them for 10 min on ice. Collect the cells by centrifugation for 5 min at 5,000 rpm.
4. Re-suspend the cells in 20 ml of ice-cold TB. Add DMSO to a final concentration of 7 % (1.4 ml) by gently shaking the Falcon tube.
5. Transfer 0.2 ml aliquots into labeled Eppendorf reaction tubes and freeze the cells in liquid nitrogen. Store the cells at -80°C.

### Transformation of *E. coli*

##### The Material

* LB medium
* LB agar plates supplemented with the appropriate antibiotics

##### The Procedure

1. Put your ligation samples on ice, defreeze 100 ml of your competent  *E. coli*  cells on top of the ice and add the cells to your ligation samples. Mix it carefully!
2. Incubate the Eppendorf reaction tubes for 30 min on ice, transfer the tubes for 90 sec to 42°C (heat shock) and put them back on ice for 5 min.
3. Add 500 ml LB medium to the cells, transfer them to 15 ml Falcon tubes (or in sterile 1.5 ml Eppendorf reaction tubes) and incubate the cell for 1 h at 37°C with agitation.
4. Propagate 50 ml of the cells on LB medium agar plates supplemented with the appropriate antibiotics. The remaining cells are collected by centrifugation for 1 min at 13,000 rpm and remove 400 ml of the supernatant. Re-suspend the pellet in the remaining 50 ml of the supernatant and propagate the cells on the same LB medium agar plates. It is highly recommended to do a negative control (only cells, no DNA).
5. Incubate the plates over night at 37°C. The plates should be stored after incubation over night at 4°C to avoid the emergence of satellite colonies.

![  ]( https://static.igem.org/mediawiki/2018/0/05/T--Goettingen--methodtrafo.png)

### Preparation and transformation of competent *B. subtilis* cells

##### The Material

* LB medium
* MN medium
* MNGE medium
* Expression mix
* Glucose (20 %)
* 1 M MgSO4
* CAA (10 %)

##### The Procedure

Preparation of competent cells

1. Inoculate 4 ml LB liquid medium with a single colony of a *B. subtilis* strain and incubate the culture over night at 28°C with agitation.
2. Use the overnight culture to inoculate 10 ml MNGE medium supplemented with 0.1 % CAA in a 100 ml shake flask to an approximate OD600 of 0.1. Incubate the culture at 37°C and 220 rpm until an OD600 of about 1.3. This may take up to 5 h, depending on the strain.
3. Dilute the culture 1:1 with pre-warmed MNGE (w/o CAA) and incubate the culture for another h at 37°C on a shaker.
    You can continue with the transformation of *B. subtilis* directly after the nutritional starvation step (see Transformation of *B. subtilis*, step 7) or continue for long term storage.
4. Transfer 15 ml of the culture to 15 ml Falcon tubes and harvest the cells by centrifugation for 5 min at 5,000 rpm. Transfer the supernatant into a sterile Falcon tube.
5. Re-suspend the cells in 1.8 ml of the supernatant, add 1.2 ml 50 % glycerine, mix the cell suspension and store the competent cell in 300 ml aliquots at -80°C.
6. Thaw an aliquot of the frozen, competent bacteria and mix 300 μl of them with 1.7 ml 1x MN medium that has been supplemented with 43 μl glucose (20 %) + 34 μl 1 M MgSO4.
7. Add 0.1 - 1 μg DNA (2 mg plasmid DNA) to 400 µl of the competent cells and incubate the reaction tube for 30 min at 37°C.
8. Add 100 μl expression mix and if required an inducer (IPTG, xylose,…).
9. Incubate the bacteria for 1 h at 37°C with agitation and propagate the cells on SP medium agar plates supplemented with the appropriate antibiotics

**Remarks: Do not forget the negative control!!!**

## Working with DNA

### Preparation of chromosomal DNA from *B. subtilis*

Here are different methods available for the isolation of chromosomal DNA. We will use the peqGOLD Bacterial DNA Kit from PEQLAB for the preparation of chromosomal DNA. The amount of DNA that will be obtained using this kit is much lower than that obtained with the standard phenol/chloroform extraction. However, the kit was designed for rapid purification of phenol- free total DNA, which is sufficient for PCR, cloning and even genome sequencing.

##### The Material

* Lysis buffer
* RNase A solution
* EtOH (100 %, ice-cold)
* peqGOLD Bacterial DNA Kit

##### The Procedure

1. Inoculation of 10 ml LB medium supplemented in a 100 ml shake flask with a single colony of  *B. subtilis*. The overnight culture has to be incubated with agitation (220 rpm) at 37°C.
2. Collect the cells from 1.5 to 2 ml of the overnight culture in Eppendorf tubes by centrifugation for 2 min at 13,000 rpm. Discard the supernatant and re-suspend the cells in 200 ml lysis buffer.
3. Incubate the mixture for 30 - 60 min at 30°C in the lysis buffer to destroy the cell wall.
4. Spin down the cells for 5 min at 4,000 g and discard the supernatant. Re-suspend the pellet in 400 µl DNA Lysis Buffer T and add 20 µl Proteinase K and 15 µl RNase A. Vortex for 10 seconds.
5. Incubate the sample for 30 min at 70°C on a shaker. Without thermo-shaker, vortex 3 times for 10 seconds in the incubation time.
6. Add 200 µl of the DNA Binding Buffer and mix gently. Add the whole sample to the column (with all the precipitants). After centrifugation for 1 min at 8,000 – 10,000 rpm, discard the flow through. Put the remaining solution into the column and repeat the centrifugation step.
7. Wash the DNA on the column with 650 ml with the DNA Wash Buffer complemented with EtOH. Each time, centrifuge for 1 min at 8,000 – 10,000 rpm and discard the flow trough.
8. Place the empty DNA column into the collection tube and centrifuge for 2 minutes at 8,000 – 10,000 rpm. This step is essential! Do not reduce the time!
9. Place the column into a new 1.5 ml Eppendorf reaction tube. To elute the DNA, add 50 ml of buffer Elution Buffer (or sterile HPLC-H2O) that has been pre-warmed to 70°C and incubate the column for 3 min at room temperature. Collect the DNA by centrifugation for 1 min at 8,000 rpm.
10. To increase the amount of chromosomal DNA, you may add a second time your first elution to the column or add again 50 µl Elution Buffer or water and centrifuge again for 1 min at full speed.

### Isolation of plasmid DNA from *B. subtilis*

The cell wall of the Gram-positive bacterium  *B. subtilis*  cannot be disrupted as easy as that of a Gram-negative bacterium like  *E. coli* . Therefore, lysozyme has to be added to the re-suspension buffer A1. Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of 1,4-b-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan.

##### The Material

* Liquid LB medium
* NucleoSpin® plasmid kit, Macherey & Nagel
* Buffer A1 supplemented with 3 mg/ml lysozyme

##### The Procedure

1. Use a single colony from your  *B. subtilis*  plate to inoculate 4 ml LB liquid medium supplemented with the appropriate antibiotic and grow the culture over night at 37°C with agitation.
2. Collect the cells from 2 ml of the overnight cultures by centrifugation for 1 min at 13,000 rpm. Re-suspend the pellet in 250 µl of buffer A1 with lysozyme und incubate for 15 min at 37°C. Add 250 ml of buffer A2, mix gently 6 – 8 times and incubate the suspension for 5 min at room temperature.
3. Add 300 ml of buffer A3, mix gently 6 – 8 times and centrifuge the lysate for 10 min at 13,000 rpm.
4. Transfer the supernatant into a NucleoSpin® plasmid column, centrifuge for 1 min at 13,000 rpm and discard the flow-through.
5. Add 500 ml buffer AW pre-heated to 50°C to the column, centrifuge for 1 min at 13,000 rpm and discard the flow-through.
6. Add 600 ml of buffer A4, centrifuge for 1 min at 13,000 rpm and discard the flow- through.
7. To dry the silica membrane, centrifuge again for 2 min at 13,000 rpm.
8. Put the column in a sterile Eppendorf reaction tube and add 50 µl of HPLC water pre-heated to 70°C on top of the silica membrane of the column, incubate the column for 5 min at room temperature and elute the DNA by centrifugation for 1 min at 13,000 rpm.
9. Quantify the plasmid DNA with the Nanodrop spectrophotometer. The DNA can be stored at -20°C.

If the concentration of the plasmid DNA is too low for sequencing (less than 50 ng/µl) you can transform *E. coli*  with the plasmid and isolate the plasmid again.

### Isolation of plasmid DNA from *E. coli*

##### The Material

* Liquid LB medium
* NucleoSpin® plasmid kit, Macherey & Nagel

##### The Procedure

1. Use single colonies from your transformation plates to inoculate 4 ml (15 – 20 ml for low-copy plasmids) LB liquid medium supplemented with the appropriate antibiotic and grow the culture over night at 37°C with agitation.
2. Collect the cells from 1.5 ml (15 ml for low-copy plasmids) of the overnight cultures by centrifugation (1 min, 13,000 rpm). Re-suspend the pellet in 250 ml of buffer A1, add 250 ml of buffer A2, mix gently 6 – 8 times and incubate the suspension for 5 min at room temperature. For the isolation of low-copy plasmids add 500 ml of buffers A1 and A2 to the cells.
3. Add 300 ml (0.6 ml for low-copy plasmids) of buffer A3, mix gently 6 – 8 times and centrifuge the lysate for 5 – 10 min at 13,000 rpm.
4. Transfer the supernatant into NucleoSpin® plasmid column, centrifuge for 1 min at 13,000 rpm and discard the flow-through.
5. (Optional: for sequencing of low-copy plasmid DNA add 500 ml buffer AW to the column, centrifuge for 1 min at 13000 rpm and discard the flow-through.)
6. Add 600 ml of buffer A4, centrifuge (1 min, 13,000 rpm) and discard the flow-through.
7. To dry the silica membrane, centrifuge again for 2 min at 13,000 rpm.
8. Add 50 ml of sterile and deionized water on top of the silica membrane of the column incubate the column for 1 - 5 min at room temperature and elute the DNA by centrifugation for 1 min at 13,000 rpm.
9. The plasmid DNA is highly pure and can be stored at –20°C.

### Concentration and purity of DNA/RNA samples

#### Determination of DNA/RNA concentration

A reliable method for the quantification of DNA and RNA is essential for our work in the lab. The concentration of nucleic acids can be determined by fluorescence and absorption spectroscopy. The quantification of nucleic acids by fluorescence spectroscopy relies on binding to fluorescent dyes, e.g. ethidium bromide. This method is very sensitive (10 – 100 ng of DNA can be detected) but time-consuming. This is the reason that DNA is mainly quantified by absorption spectroscopy. DNA can be quantified by absorption spectroscopy because it absorbs ultra violet (UV) light with a maximum at the wavelength of 260 nm. However, RNA absorbs UV light also at 260 nm and aromatic amino acids at 280 nm what means that both can contaminate your measurements. Therefore, it is important to determine the purity of your samples.

#### The purity of the DNA preparation

The quantification of DNA makes only sense if the sample is pure. The purity of the sample can be evaluated by recording a spectrum of your DNA solution in the range between 220 and 320 nm. Proteins in the sample absorb light at 280 nm and cause a reduction of the A260 nm/A280 nm ratio. If the DNA sample is pure, this ratio is in the range of 1.8 – 2.0. A strong peak at 230 nm can indicate contamination with organic compounds or chaotropic salts.

##### The Material

* Nanodrop ND-1000 Spectrophotometer
* DNA/RNA sample
* Sterile water

##### The Procedure

1. Clean the measurement pedestal (it contains the receiving fibre) with deionized water and pipette 1 ml of water on top of it.
2. Close the lid (sampling arm). Now the upper measurement pedestal (it contains the second fibre optic cable) is brought into contact with the liquid sample causing the liquid to bridge the gap between the two fibre optic ends. The gap is controlled to both 1 and 0.2 mm paths.
3. Initiate a spectral measurement using the operating software on the connected PC by pressing the “blank” button to blank the Nanodrop. A pulsed xenon flash lamp will provide the light source and the spectrophotometer analysis the light after passing through the sample. After the measurement the data is logged in an archive file on the PC.
4. When the blanking is done, open the sampling arm and wipe the sample from both the upper and lower pedestals using a soft laboratory wipe. Wiping prevents sample carryover in successive measurements for samples varying by more than 1,000-fold in concentration.
5. Pipette 1 ml your DNA sample and press the “Measure” button to determine the amount of DNA in your sample.
6. Remove the DNA solution from the measurement pedestal and clean the lid and the pedestal with deionized water.

### Agarose gel electrophoresis

Linear and double-stranded DNA molecules can move through a gel matrix with a velocity that is proportional to the logarithm of their molecular weight (MW). Therefore, the MW of unknown DNA species can be determined by comparing their electrophoretic mobility with that of DNA molecules having known MWs. DNA molecules with lengths ranging from 0.5 kbp to 25 kbp can be separated using agarose gels.

##### Voltage

The velocity with that DNA molecules move through a gel matrix depends on the applied voltage. It is important to know that the velocity of large DNA molecules increases more rapidly than that of small DNA molecules. As a consequence, large molecules can be separated from each other with a high accuracy at low voltages.

##### The electrophoresis buffer

Most gel systems are based on Tris-Acetate-EDTA (TAE) and Tris-Borat-EDTA (TBE) buffers. The electrophoretic mobility of DNA molecules is quite similar in both buffers. However, the buffering capacity of the TBE buffer is significantly higher. Therefore, it is recommended to use TBE buffer for mini-gels that run at a high voltage.

##### The DNA conformation

Supercoiled, nicked and linear DNA molecules, having the same molecular mass move with different velocities through the gel matrix. Because supercoiled DNA has the smallest hydrodynamic radius, these DNA species show the highest electrophoretic mobility.

##### The Material

* 1x TAE buffer or 1x TBE
* 5x DNA loading dye
* Agarose
* HDGreen® Plus Safe DNA Dye or Midori Green

##### The Procedure

1. Prepare a 100 ml mixture of 1 % agarose in 1x TAE. Carefully heat the mixture in a microwave to dissolve the agarose. You can either store the agarose solution at 65°C or cool it down to room temperature and heat it again for the next gel.
2. Now it is important to wear gloves. Add 3 µl of the HDGreen® Plus Safe DNA Dye to 30 ml dissolved agarose and pour the gel in a small custom-made gel chamber.
3. While the gel is cooling down, prepare your DNA samples by mixing 2 – 5 µl of your DNA solution with 0.4 – 1 µl of loading dye, either in Eppendorf reaction tubes or in a multi- well microtitre plate.
4. Remove the comb from the cold gel, add 1x TAE/TBE buffer into the chamber and load the slots with your DNA samples. Do not forget the size standard (λ-marker or commercial 1 kb DNA ladder).
5. Connect the gel chamber with the power supply and switch it on. Adjust the voltage to 100 – 130 V and press the start button.
6. If the stain bromphenol blue has passed ¾ of the gel, you should switch off the power supply and check your gel under UV light. Safe picture and print result for lab book.
7. If possible, take a picture for the group seminar and your lab notebook.
8. You can also stain your gel after the run has finished in a 1 % aqeous HDGreen® Plus Safe DNA Dye or Midori Green solution for 10 - 30 min at room temperature.

##### Ethidium bromide staining

To stain a agarose gel with Ethidium bromide you have to replace step two and stain the gel after running with the following procedure:

1. Put on one glove and transfer the gel into the staining solution (0.5 µg/ml of Ethidium bromide in water). After 10 min you can destain the gel in the water bath.
2. After 10 – 30 minutes of destaining you may analyze your agarose gel, either on a UV table or with a gel documentation device (imager).

##### Error sources

###### The DNA molecules were separated with low accuracy

Wrong agarose concentration; low voltage may cause diffusion of DNA molecules due to a long run.

###### Smearing bands

Too much DNA has been loaded to the slots; the applied voltage has been too high. The gel looks smeared at all, you might touched the gel directly with your hands.

###### The gel was melting

Even old lab veterans sometimes use water instead of 1-fold TAE to dissolve agarose. You should also regularly refresh the TAE buffer inside the running chamber.

##### Further information concerning the HDGreen® Plus Safe DNA Dye

###### Security precaution

Ethidium bromide is commonly used to stain nucleic acids in molecular biology laboratories. However, this agent is highly mutagen and therefore considered as hazardous. As alternative staining method we use HDGreen® Plus Safe DNA Dye. According to the manufacturer (INTAS) this dye does not contain substances which at their given concentration, are considered to be hazardous to health. Nevertheless, this dye stains DNA and every DNA staining substance should be treated carefully and only with gloves.

###### Disposal considerations

The waste must be disposed of in accordance with federal, state and local environmental control. Therefore, the gels are collected in a container next to the imager.

### Purification of DNA fragments

#### PCR purification kit

##### The Material

* DNA product(s) or linearized plasmid(s)
* peqGOLD Cycle-Pure Kit

##### The Procedure

1. Add an equal volume of CP Buffer to your DNA solution. For fragments < 200 bp add 3 volumes of CP Buffer for better results.
2. Transfer the mixture to a spin column and centrifuge for 1 min at 13,000 rpm. Discard the flow-through and add 750 µl CG Wash Buffer.
3. Centrifuge for 1 min at 13,000 rpm, discard the flow-through and repeat the steps 2 and 3 once.
4. Centrifuge again to dry the silica membrane 2 minutes at 13,000 rpm.
5. Put the column into a labeled Eppendorf reaction tube, add 30 - 50 µl of water (not less than 30 µl) on top of the silica membrane, incubate for 5 min at room temperature and elute the DNA by centrifugation for 1 min at 13,000 rpm. Please keep in mind that you have to add less water if you want to elute the DNA from a restriction analysis! Always check the purified DNA by agarose gel electrophoresis to make sure that you did not lose the DNA. Determine the DNA concentration and the purity.

##### Concentration of DNA samples

You can concentrate your DNA sample if the amount is too low. Therefore add 1 M NaCl to a final concentration of 0.1 M and add two volumes of 100 % EtOH. Vortex your samples and spin down for 15 minutes at 13,000 rpm. The supernatant can be discarded. Add then 700 µl 70 %age EtOH and centrifuge again for 2 min at 13,000 rpm. Let the EtOH evaporate at room temperature. Now you can dissolve your pellet in water.

### How to design primers

One of the major tools in molecular biology is the polymerase chain reaction (PCR) where DNA fragments are amplified in a logarithmic manner. Therefore, you need at least two primers that bind to your template DNA strands and boarder the region that should be amplified. It is obvious that good primer design is essential for successful amplification.

##### The Material

* Template sequence + the sequence of the flanking regions
* Program for primer design (optional)

##### The Procedure

The optimal length of primers should be around 18 - 30 bp and the melting temperature should be around 52 - 64°C (usually 58 - 62°C). The temperature can be calculated with a program (e.g. Oligo Calc) or manually (for each C or G add 4°C, for each A or T add 2°C). Non-binding sites are not calculated in the melting temperature (like flanks or additional restriction sites).

Typical set-up for a forward and a reverse primer. The two primers anneal within the PCR reaction to the two complementary strands. The reading direction is different so that they produce a product. For sequencing the primer can also read only in one direction. The primer should bind once so the template (plasmid or chromosomal DNA). The GC content should be around 40 - 60 % to provide a stronger binding to the template. It is necessary to check if the primers can form undesired secondary structures that can lead to the disturbance of the PCR. This and the self- complementarities can also be analyzed by programs, like Oligo Clac.

For each method the primers need to be designed specifically. It has to be considered if the part of the DNA that will be amplified via PCR needs to be in frame in the further experiments. In this case a few bp should be added to assure the right reading frame. For some experiments it might also be necessary to remove the stop codon of the gene of interest. A crate of beer is demanded for every wrong designed AND ordered prime.

![Selfhtml](https://static.igem.org/mediawiki/2018/d/d9/T--Goettingen--methodprimer.png)

### The polymerase chain reaction (PCR)

Although the basic principle of the polymerase chain reaction (PCR) has been described already in 1985, this simple method is still very important in molecular biology (Sakai et al., 1985). You just need a few things to set up a PCR: template DNA, oligonucleotides that hybridize to the template, a thermostable polymerase, buffer and nucleotides.

##### The Material

* PhuS polymerase or *Taq* polymerase, purified in-house
* Template DNA (chromosomal, plasmid DNA)
* 5x HF or GC buffer
* 12.5 mM dNTPs
* 5 pmol forward primer
* 5 pmol reverse primer

##### The Procedure

Prepare for your sample a reaction mixture according to the scheme below. If you have many reactions prepare a master mix and scale up the reaction volumes. For many applications a 50 µl sample is sufficient. For fragments that should be used for cloning use commercial Phusion polymerase, this is less error prone.

Pipetting scheme for a 100 µl *PfuS* sample

| Compound | Volume in µl |
| --- | --- |
| 5x HF buffer | 20 |
| dNTPs | 4 |
| forward primer | 4 |
| reverse primer | 4 |
| Template DNA (chromosomal or other) | 1–2 |
| DNA polymerase ( *Phusion* or *PfuS*) | 1 |
| sterile H2O | 66–67 |

Pipetting scheme for a 100 µl *Taq* sample

| Compound | Volume in µl |
| --- | --- |
| 10x Taq buffer | 10 |
| MgCl2 | 6 |
| dNTPs | 1 |
| forward oligo | 4 |
| reverse oligo | 4 |
| Template DNA (chromosomal or other) | 1–2 |
| Taq polymerase | 1 |
| sterile H2O | 72–73 |

##### Troubleshooting for PCR

Try to do the following steps to solve your PCR problem:

* Dilute your primers anew, to be sure the primers are the correct ones. This helps in most cases when the PCR does not function at all.
* Dilute your template, to lower the template concentration and enhance the PCR efficiency.
* Longer elongation time.
* Longer denaturation time.

### Digestion of DNA

Digestion of DNA by restriction endonucleases is an important method in molecular biology. Restriction enzymes recognize 4 – 10 bp-long palindromic DNA sequences in a highly specific manner. For instance, the enzyme  *EcoRI*  from the *Escherichia coli* strain R recognizes the sequence 5’-GAATTC-3’. In the early days, the reaction samples to digest DNA were incubated for up to 20 h in order to obtain a fully digested DNA. Nowadays, many biotech companies sell hyperactive variants of the restriction enzymes that are commonly used in the lab. We will use FastDigest enzymes from Thermo Scientific. According to the manual from the manufacturer, 1 mg of DNA (for instance l phage DNA) are cleaved by 1 ml of the FastDigest enzyme within 5 – 15 min. With the same enzyme activity, 1/5 of a purified PCR product should be cleaved within 20 min.

##### The Material

* DNA sample
* Restriction enzyme
* 10x Buffer

##### The Procedure

Reaction mixture to digest DNA

| Compound | Volume in µl |
| --- | --- |
| 10x buffer | 4 |
| Enzyme | 3 |
| Plasmid DNA (125 ng/µl) | 8 |
| sterile H2O | add 40 |

We recommend incubating the reaction mixtures for at least 30 min at 37°C to obtain fully digested DNA fragments. After digestion, you can use the PCR purification kit from Qiagen to purify the digested DNA. Please keep in mind that you have diluted the DNA solution in the reaction mixture. For eluting the DNA from the spin column you should use 28 – 30 ml of sterile water. To dephosphorylate your plasmid DNA, add 1 ml alkaline phosphatase to the reaction mixture and incubate the plasmid for additional 15 min at 37°C. Watch out for star activity of the restriction enzyme in your sample (e.g. *EcoRI*)! Star activity depends on the enzyme amount, the pH value, the presence of organic solvents, and the incubation time (Wei et al., 2008).

##### There are different types of restriction enzymes:

The types of restriction enzymes

| Type | Characteristics | Example |
| --- | --- | --- |
| I | 3 subunits: S recognizes a specific sequence M methylates and R cuts the DNA unspecifically | *EcoKI* |
| II | 2 subunits: a methylase and a restriction enzyme;  both enzymes recognize the same DNA sequence | *BamHI* |
| III | Several subunits: these enzymes cut a specific  sequence 20-25 away from the recognition site | *EcoPI* |

### Ligation of DNA

Ligation is the process in which a digested plasmid is covalently connected to an insert (PCR product), which has been digested with the same restriction enzyme(s). If both, the insert and the plasmid, had been digested with only one enzyme, it is strongly recommended to dephosphorylate the plasmid prior to mix the ligation sample. Otherwise it takes your whole Master thesis or Ph. D. thesis until you find a plasmid containing the insert. We are using the T4 DNA ligase, which is a standard enzyme for ligation. In contrast to the NAD+-dependent DNA ligase, which needs NAD+ as a cofactor, the T4 DNA ligase uses ATP. Moreover, while the NAD+- dependent DNA ligase can connect only DNA fragments with sticky ends, the T4 DNA ligase can ligate sticky and blunt ends.

##### The Material

* T4 DNA ligase
* 10x Ligation buffer (keep aliquots on ice, contains ATP)

##### The Procedure

1. Defreeze an aliquot of the ligation buffer on ice (it contains ATP) and mix the ligation and re-ligation samples in a 1.5 ml Eppendorf reaction tube:

   Reaction mixture for ligation and re-ligation

   | Ligation | Re-ligation | Compound |
   | --- | --- | --- |
   | sample (µl) | sample (µl) |  |
   | 1 | 1 | T4 DNA ligase |
   | 2 | 2 | 10x Ligation buffer |
   | 3 | 0 | 150 ng Insert |
   | 1 | 1 | 50 ng Plasmid |
   | 13 | 16 | sterile H2O |
2. Incubate the samples for at least 2 h at room temperature or over night at 16°C.
3. Use the complete ligation and re-ligation samples for transformation of competent
   *E. coli* cells. You should include a positive control (circular plasmid DNA) and a negative control (no DNA) to ensure that your  *E. coli* cells were competent and not contaminated, respectively. Moreover, the ligation/re-ligation ratio will tell you whether it is worth to analyze your clones.

##### Error sources

###### No transformants at all on the plates:

The ATP in the ligation buffer was hydrolyzed (old buffer). Probably you forget a compound in the ligation mixture. The cells were not competent.

###### Same number of ligands and re-ligands on the plates:

Probably your DNA was not completely digested. Therefore, it is very important to check you digested DNAs by agarose gel electrophoresis prior to the ligation. You can at a least see that your plasmid DNA was linearized.

### 3A Assembly

1. Restriction Digests

1. The left part sample is cut out with *Eco*RI and *Spe*I.
2. The right part sample is cut out with *Xba*I and *Pst*I.
3. The linearized plasmid backbone is a linear piece of DNA. It has a few bases beyond the *Eco*RI and
   *Pst*I restriction sites. It is cut with *Eco*RI and *Pst*I.

2. All 3 restriction digests are heated to heat kill all of the restriction enzymes.
3. An equimolar quantity of all 3 restriction digest products are combined in a ligation reaction.
4. The desired result is the left part sample's *Spe*I overhang ligated with the right part sample's
   *Xba*I overhang resulting in a scar that cannot be cut with any of our enzymes.
5. The new composite part sample is ligated into the construction plasmid backbone at the *Eco*RI and
   *Pst*I sites.- When the ligation is transformed into cells and grown on plates with antibiotic chloramphenicol, only colonies
     with the correct construction survive.

![](https://static.igem.org/mediawiki/parts/4/42/3AAssembly.png)

3A Assembly overview.

### DNA Sequencing

The correct sequences of PCR products or of newly constructed plasmids having the desired insert can be confirmed by the in-house sequencing facility, the G2L or by sending the DNA to the companies SeqLab (Göttingen). All facilities determine the DNA sequence by the chain- termination method that has been developed by Frederick Sanger in 1977. Sanger received in 1980, together with Paul Berg and Walter Gilbert, the Nobel Prize in chemistry. This became his second Nobel price because Sanger had been already awarded in 1958 for his work on protein sequencing. The Sanger method is based on the amplification of the template by a DNA polymerase, and the incorporation of normal deoxynucleotidetriphosphates (dNTPs) and modified dideoxynucleotidetriphosphates (ddNTPs). The incorporation of modified (fluorescently-labeled) ddNTPs results in termination of strand elongation. Sequencing with fluorescently-labeled ddNTPs permits DNA sequencing in a single reaction and the fast analysis of the DNA fragments by an optical read out system.

![](https://static.igem.org/mediawiki/2018/f/f2/T--Goettingen--methodsequenzing.png)

##### The Procedure

###### …for the [G2L:](http://appmibio.uni-goettingen.de/index.php?sec=g2l)

You need a personal folder on the network drive from the G2L. The contact person at the G2L is Dr. Sonja Voget. Her phone number is 33655. This folder contains a template Excel sheet, which you can fill in (sample labels, amount of samples etc.). Label your tubes starting with “NameSurname\_1… NameSurname\_n + 1”. Send the Excel sheet to “sequence@g2l.bio.uni-goettingen.de”. The folder also contains a Word document, which you have to fill in (again sample labels, amount of samples etc.). Print out the form and bring it together with your samples to the G2L until 15:00 o’clock on Friday afternoon. You will find the sequencing data in your personal folder usually at the following Wednesday.

###### …for [Seqlab:](https://www.microsynth.seqlab.de/home-de.html)

Label your tubes with the pre-printed barcodes. Type Login to the SeqLab homepage and select “Economy Run”, type in your mail address and select the number of samples you have prepared for sequencing. Next you have to choose the number that has been used to label your first sample and finally confirm your order. Put all the samples in a plastic bag and the printed sheet (ordering details and number). Put this bag till 6 p.m. into the post box, which is located at the entrance at the back of the building. Sequencing by SeqLab is performed over night. You will get your sequences next day.

Overview of sample preparation

| Sequencing service | G2L | SeqLab |
| --- | --- | --- |
| DNA (ng) | 200 | 600 (PCR product) to 1,200 (plasmid) |
| Oligo (µl, pM) | (1, 5) | (1, 20) |
| Final volume (µl) | 5 | 15 |
| Reaction tube (ml) | 1.5 | 1.5 |

##### Evaluation of your sequencing data

You can analyze your sequencing data using the Geneous which is installed on a PC in our department. For further information about the amino acids have a look to 105. Amino acids.

### Combined-chain reaction (CCR) and multiple-mutation reaction (MMR)

![](https://static.igem.org/mediawiki/2018/f/f6/T--Goettingen--methodmmr.png)

Several methods for PCR-based site-directed mutagenesis have been developed. Among these, the combined-chain reaction method proved to be very rapid and reliable (Bi & Stambrook, 1997). The principle of this method is the use of mutagenic primers that hybridize more strongly to the template than the external primers. The mutagenic primers are phosphorylated at their 5’ ends, and these are ligated to the 3’-OH groups of the extended upstream primers by the action of a thermostable DNA ligase (see the Figure beside for the principle). Moreover, the DNA polymerase employed must not exhibit 5’ 3’ exonuclease activity, to prevent the degradation of the extended primers. In our view, Pfu and Pwo polymerases are both well suited. The original protocol describes the introduction of two mutations simultaneously. In a previous study, we used a combined chain reaction to mutagenize four distant bases in a DNA fragment in a one-step reaction.

The MMR is a further development of the CCR and allows the simultaneous introduction of up to nine mutations in a single PCR approach (more have yet to be proven; Hames et al., 2005). The MMR was especially designed for the work with Mycoplasma genes in order to express recombinant genes in *E. coli* . The species Mycoplasma differ in their genetic code and use the opal codon (UGA) to introduce tryptophan instead of terminating the translation. Hence, it is absolutely necessary to exchange the opal codons, if present, with UGG codons (TGG on DNA level). Meanwhile, the in vitro synthesis of oligonucleotides is getting more inexpensive and will replace this method in the future, especially, because the MMR can be very time consuming.

##### The Material

* 10x CCR/MMR buffer
* Phusion or AccuzymeTM polymerase
* 5 U/ml Ampligase
* Primer for amplification
* 12.5 mM dNTPs
* Mutagenesis primer
* BSA (20 mg/ml)

##### The Procedure

Before you start your CCR or MMR you should amplify your gene of interest and clone it into a plasmid of your choice. The introduction of mutations is far more efficient when including this step.

CCR/MMR reaction setup

| Compound | Volume in µl |
| --- | --- |
| 20 pmol forward primer | 2 |
| 20 pmol reverse primer | 2 |
| mutagenesis primer | 4 |
| Plasmid DNA | 1 |
| 10x CCR buffer or HF buffer | 5 |
| Accuzyme™ or Phusion polymerase | 1 |
| Ampligase | 3 |
| dNTPs | 2 |
| BSA (20 mg/ml) | 2 |
| sterile H2O | 30 |

Do not forget to adjust the elongation time according to your polymerase and the length of the template.

### Overexpression of recombinant proteins

Large-scale purification is necessary to achieve enough protein for further experiments, e.g. crystallization or enzyme activity assays. After the evaluation of the best expression conditions with test expressions you can scale up your samples.

##### The Material

* LB liquid medium (e.g. 1 liter per protein,)
* 1 M Isopropyl-D-thio-galactopyranoside (IPTG) stock
* Washing buffer (e.g. buffer W,)
* Elution buffer (e.g. buffer E,)

##### The Procedure

1. Inoculate an overnight culture in 25 - 100 ml LB medium with the desired strain(s) and antibiotics. Incubate the culture at 37°C and 220 rpm over night.
2. Inoculate the over day cultures (usually 500 ml - 1 l in a 2 l baffled flask with long neck) to an OD600 of 0.1 with your overnight culture and add the appropriate antibiotic. Grow the cultures at 37°C with agitation to an OD600 of 0.6 - 0.8. Then add IPTG to a final concentration of 1 mM. Now the expression of the heterologous gene is induced.
3. After induction, incubate the cultures for additional 2 - 3 h at 37°C with shaking (if these are the conditions of choice).
4. Split the cultures into two 500 ml samples and centrifuge (15 min, 5,000 rpm) at 4°C.
5. Discard the supernatant (in case your protein is not secreted).
6. Re-suspend the pellet in 15 ml buffer W, transfer it to a 50 ml falcon tube and centrifuge the tubes at 8,500 rpm and 4°C for 10 - 15 min.
7. Discard the supernatant and freeze your cell pellet (-20°C) or continue with cell disruption.
8. Re-suspend each pellet in 25 - 50 ml of washing buffer. Now your samples are ready for cell disruption, e.g. using the French Press method.

### Preparation of cell-free crude extracts

This method can be applied to detect proteins, which are really strongly produced by the bacteria. The cell-free crude extract that has been obtained by this procedure can also be used for Western blotting. A drawback of this method is that you may lose some protein by proteolysis. Moreover, the amount of protein you will get is sometimes quite low.

### The Material

* 1x PBS, pH 7.4
* Lysis buffer
* (recommended to add protease inhibitor: EDTA, Pefabloc, PMSF)

### The Procedure

1. Harvest 2 ml from a  *B. subtilis*  culture that has been grown over night or to an OD600 of approximately 1 - 2 by centrifugation for 2 min at full speed.
2. Re-suspend the cells in 50 µl lysis buffer. Keep some ml of the lysis buffer for the Bradford assay. Incubate the cells for 30 min at 37°C and 350 rpm on an Eppendorf thermomixer.
3. Separate the soluble proteins from the cell debris by centrifugation for 10 min at full speed and 4°C. Transfer the supernatants into new Eppendorf reaction tubes. Determine the protein concentration and analyze your samples by SDS PAGE and Western blotting.

### Cell disruption by French press

Many experiments required the disruption of bacterial cells, e.g. to purify proteins for enzyme activity assays or protein-protein interaction studies. A widely used method in our lab for cell disruption is using a French pressure cell, or simply French press. In principle, a French press disrupts the cell wall and plasma membrane by passing the cells through a narrow valve under high pressure. The name has nothing to do with the machine some people prepare their coffee in, but was named after its inventor Charles Stacy French of the Carnegie Institution of Washington.

##### The Material

* French press (small or big)
* Disruption cell (bomb) Pre-cooled in a water bath or in the fridge
* Re-suspension buffer Pre-cooled in the fridge or on ice
* Cell pellets Frozen cells within a 50 ml falcon tube

##### The Procedure

**All steps must be carried out on ice!**

1. Re-suspend your cells pellet in re-suspension buffer by vortexing or pipetting up and down. Usually we use a pellet from 500 ml of cells with an OD600 of 0.8 – 1.2 and re-suspend it in 15 ml cold buffer of choice.
2. Turn on the French Press and select the pressure for cell disruption (18,000 psi for
    *B. subtilis*  and  *E. coli* ).
3. Fill the re-suspended cells in the disruption cell and close it tightly.
4. Put the closed disruption cell into the machine so that the piston is on the top. Make sure that the handles of the piston are turned in your direction so they cannot come in contact with the mounting while the cell gets compressed.
5. Press start and follow the instructions on the display of the machine.
6. Open the valve slowly while you put a 50 ml falcon under it.
7. Collect the opened cells that flow out of the valve. Watch out that the cell suspension flows out slowly and the pressure remains almost constant until the whole suspension has flown out.
8. Reload the disruption cell. For Gram-positive bacteria, open up the cells three times. For Gram-negative cells one run should be enough.
9. Spin down your disrupted cells at 8,000 rpm for 15 min at 4°C. Proceed with the supernatant.
10. Fill the supernatant into ultracentrifugation tubes and balance tem. Spin down for 1 hour at 4°C and 35,000 rpm. Tubes must be filled with buffer at least with half of the tube-volume.
11. In the meantime, clean the disruption cell with ethanol (70 %) and distilled water. Then store it in the fridge until next use.
12. Fill the supernatant into pre-cooled falcon tube.

### Purification of Strep-tagged proteins

The Strep-tag II is a short peptide (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin®, an engineered streptavidin. The binding affinity of Strep-tag II to Strep-Tactin® (KD = 1 mM) is nearly 100 times higher than to streptavidin. This technology allows one-step purification of almost any recombinant protein under physiological conditions, thus preserving its bioactivity.

##### The Material

* Buffer W
* Buffer E
* Crude extracts of your sample cells harboring the desired protein
* Strep-Tactin® sepharose (50 %; 1 ml per 1 l cell culture)

##### The Procedure

###### Equilibration of the column

1. Remove the bottom cap from the column and apply the appropriate amount of Strep- Tactin® matrix (1 ml of the 50 % suspension per 1 l of cell culture, column bed volume = 0.5). For all steps, allow the solutions on the column to drain off by gravity before adding the next one.
2. Equilibrate the column by adding 10 CV (CV = column bed volume) of buffer W.

###### Adsorption of Strep-tag proteins to the matrix and column washing

1. Load your crude extract on the column and keep 50 - 100 µl as a sample for later analysis (CE). Also, collect the flow through as a sample (FT).
2. Wash the column by adding 5 times with 2.5 CV of buffer W. Collect every fraction as “W1” - “W5”.

###### Elution of Strep-tag proteins from the matrix

1. Add 4 times 0.5 CV of buffer E and collect all fractions as “E1” - “E4”.

###### Analysis

1. To analyze the purification result, perform SDS-PAGE. For the gel, choose an appropriate polyacrylamide concentration according to the molecular weight of the purified protein. Load 5 µg of CE and FT fractions (determined by the Bradford assay) onto the gel and 15 µl of wash and elution fractions. Stain the gel with Coomassie Brilliant blue.

###### Dialysis/buffer exchange

1. To get rid of the desthiobiotin in the protein solution or to exchange the buffer, perform dialysis with the protein fraction of interest. Dialyze o/n against 1,000x volume of buffer W or a desired buffer.

###### Regeneration of Strep-Tactin column

1. Wash the column 3 times with 5 CVs Buffer R. The color should change from yellow to red.
2. The regeneration process is complete when the whole column got the same intensity in red color. When not, add more Buffer R.
3. Overlay with 2 ml Buffer W and store the column at 4°C.
4. Prior to the next use remove Buffer R by washing with 2 times 4 CVs Buffer W.

### Bradford assay

The Bradford reagent can be used to determine the concentration of proteins in solution (Bradford, 1976). The procedure is based on the formation of a complex between the dye Coomassie brilliant blue G-250 and proteins in solution. The protein-dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The absorption is proportional to the amount of protein present in the sample. The assay is suitable for a protein concentration in the range between 0.1 - 1.4 mg/ml of protein using bovine serum albumin (BSA) as the standard protein. Protein solutions or crude extracts with protein concentrations above 1.4 mg/ml have to be diluted to be in the linear range of the assay. The Bradford assay is advantageous because it is fast, the staining does not depend on the protein and the reagent is commercially available.

##### The Material

* Bradford solution, 5-fold concentrate
* Buffer from protein elution

##### The Procedure

1. Prepare for each protein mixture 3 samples with different concentrations. If you have many samples to measure, we suggest to do a master mix and distribute 1 ml of the master mix to labeled Eppendorf reaction tubes. Using a master mix, you can measure more samples at the same time and it helps you to avoid errors.

   | Sample | Reference Sample |
   | --- | --- |
   | 800 ml H2O | 800 ml H2O |
   | 0.5, 1.0 & 2.0 ml crude extract | no protein but equal volume of buffer |
   | 200 ml Bradford solution | 200 ml Bradford solution |
2. After mixing the samples with the protein solutions, the mixtures have to be incubated for 5 minutes at room temperature. Transfer the mixtures and the reference sample to 1.5 ml cuvettes and measure the absorption at a wavelength of 595 nm.

![](https://static.igem.org/mediawiki/2018/1/19/T--Goettingen--methodBSA.png)

3. Next you can calculate the amount of protein in your sample using the following formula:

   ![](https://static.igem.org/mediawiki/2018/1/1c/T--Goettingen--experiments-bredford_formula.png)

   The slope “a” of the calibration curve has been determined using the protein BSA. After several repetitions, this value turned out to be 0.0536 when 1 ml (with c = 1 µg/µl BSA) has been added to 1 ml of the five-fold diluted Bradford solution. Do not forget to subtract the absorption of your blank from the absorption of your sample!

For the ß-Gal assay, add 20 ml of your crude extract to the Bradford-water solution. Use 20 ml of the Z-buffer/LD-Mix as a reference for the measurement. The determined absorption can be directly used together with the formula for the calculation of the Miller units.

### SDS PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used method to separate complex mixtures of proteins. In an electric field the negatively charged proteins migrate towards the anode. Moving through the small pores of the gel matrix, the proteins are separated according to their size. Using a marker, the relative molecular weights of proteins within the sample can be determined. Denaturating gels are prepared as described by Laemmli et al. (1970). The gels consist of a stacking and a running gel and are poured to a thickness of 1 mm. The acrylamide in the mixtures builds long chains that are bridged by N, N′- methylenbisacrylamid (mixture 37.5:1), the result is a gel matrix. The polymerization process is induced by radical starter APS and the catalysator TEMED.

![](https://static.igem.org/mediawiki/2018/0/0a/T--Goettingen--methodSDS.png )

##### The Material

* 6x SDS loading dye
* 10x Running (PAGE) buffer
* 1.5 M Tris-HCl, pH 8.8
* 1.5 M Tris-HCl, pH 6.8
* TEMED/TMEDA (Tetramethylethylenediamine)
* Acrylamide:Bis-Acrylamide (37.5:1, TOXIC!!!)
* APS (Ammonium persulfate, 10 %)
* SDS (Sodium dodecyl sulfate, 10 %)
* Power supply & SDS PAGE devices
* Iso-propanol (100 %)

##### The Procedure

1. Clean the glass plates, combs and mats with ethanol and assemble the glass plate sandwich. Be sure that the plates are well aligned. Place the plates in the casting frame and tighten them. Place the casting frames into the casting stands.
2. Wear gloves for making the gels because acrylamide is a neurotoxin in its unpolymerized form!!!

   Running gel for denaturing SDS PAGE (enough for 2 gels)

   | Components for gel | 15% | 12% | 10% |
   | --- | --- | --- | --- |
   | H2O | 2.3 ml | 3.3 ml | 4.0 ml |
   | Acryl-Bisacrylamide 30% | 5.0 ml | 4.0 ml | 3.3 ml |
   | 1.5 M Tris-HCl, pH 8.8 | 2.5 ml | 2.5 ml | 2.5 ml |
   | SDS solution 10% | 0.1 ml | 0.1 ml | 0.1 ml |
   | APS 10% | 0.1 ml | 0.1 ml | 0.1 ml |
   | TEMED | 8 µl | 8 µl | 8 µl |

   Stacking gel for denaturing SDS PAGE (5%, enough for two gels)

   | Components for gel | 5 % |
   | --- | --- |
   | H2O | 6.83 ml |
   | Acryl-Bisacrylamide 30% | 1.3 ml |
   | 1.5 M Tris-HCl, pH 6.8 | 0.87 ml |
   | SDS solution 10% | 0.1 ml |
   | APS 10% | 0.1 ml |
   | TEMED | 10 µl |
3. Mix the ingredients of the running gel and pour the solution quickly (gel starts to polymerize once TEMED has been added) into the gap between the glass plates. Leave enough room for the stacking gel – appr. 1 cm below the bottom of the comb´s teeth. After a minute you can overlay the solution with iso-propanol or ethanol. This excludes the access of oxygen and plane the surface.
4. After polymerization, pour off the iso-propanol and rinse with distilled water. Mix the reagents for the stacking gel and pour it on top of the running gel till the space is completely full. Then insert the comb. No alcohol overlay is needed due to the comb.
5. After polymerization clamp the gel into the electrophoresis apparatus and fill it with running buffer. Remove the comb carefully and use a pipette or syringe to wash the wells with running buffer.
6. The protein samples (up to 15 µl) are mixed with SDS loading buffer (2.5 µl) and incubated for 10 min at 95°C.
7. After heating briefly centrifuge the samples to spin down the condensed water.
8. Then you can apply the samples on a SDS PAGE with a pipette or Hamilton microliter syringe. Do not forget to add molecular weight marker in one lane.
9. Run the gel at 80 V until the front has moved into the running gel. Then you can increase the voltage to 120 V. Run the gel until the running front has reached the bottom of the running gel.
10. Pry the plates apart by using a gel spacer. The gel can then be stained (Coomassie staining, or Silver staining) to visualize the separated proteins or processed further (Western Blotting).

### Coomassie staining

After separation of proteins by gel electrophoresis, the proteins can be fixed and visualized by different staining procedures. The Coomassie Brilliant blue-staining procedure is a fast and very easy method that is commonly used to detect proteins. Coomassie Brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G- 250 differs from Coomassie Brilliant blue R-250 by the addition of two methyl groups. The name "Coomassie " is a registered trademark of Imperial Chemical Industries.

![](https://static.igem.org/mediawiki/2018/8/8d/T--Goettingen--methodcoomassie.png)

##### The Material

* Fixation solution
* Staining solution
* De-staining solution

##### The Procedure

1. For staining, place the gel in a plastic container and cover the gel with fixation solution. Shake the gel for 10 min at room temperature. You can also stain the membrane after doing a Western Blot with this technique.
2. Remove the fixation solution and cover the gel with staining solution. Incubate the gel for 10 – 20 min at room temperature on a shaker.
3. Remove the staining solution (it can be re-used). De-stain the gel over night with 10 % acetic acid under rigorous shaking at room temperature. Adding a paper towel to the solution speeds up the de-staining process. Boiling the gel in a microwave speeds up de- staining.

### Silver staining

Silver staining is a very sensitive method for staining polyacrylamide gels (sensitivity 5 - 30 ng/protein band). It is widely use to check the purity of protein extracts and to identify protein-protein interactions. During staining, silver ions build up complexes with glutamate, aspartate and cysteine amino acid residues of the proteins and thereby get reduced to metallic silver. Thus, the intensity of the silver staining depends on the amino acid sequence of the different proteins and can vary considerably. Moreover, in contrast to Coomassie staining, silver staining is not appropriate for protein quantification (Winkler et al. 2007).

##### The Material

* FA-Fixation solution
* Developer
* Impregnator
* Thiosulfate solution
* Stop solution
* Ethanol (50%)

##### The Procedure

The protein bands are stained according to method of Nesterenko et al. (1994). For that purpose, the polyacrylamide gels were incubated on a shaker with the following reagents and in the stated order.

| Step | Solution | Time |
| --- | --- | --- |
| Fixing | Fixation solution | 1–24 h |
| Washing | EtOH (50%) | 20 min, 3 times |
| Reduction | Thiosulfate solution | 1.5 min |
| Washing | deion. H2O | 20 s, 3 times |
| Staining | Impregnator | 15–25 min |
| Washing | deion. H2O | 20 s, 3 times |
| Development | Developer | until sufficient staining |
| Washing | deion. H2O | 20 s, 2 times |
| Stop | Stop solution | 5 min |

### Determination of β-galactosidase activity

The “ß-Gal” assay is a very common method to determine the activity of a promoter in vivo (Miller, 1972). To monitor the promoter activity, the promoter of interest has to be fused to the lacZ gene by a simple cloning step. In the next step, the so-called promoter- reporter gene fusion has to be introduced into the chromosome of the  *B. subtilis* strain of interest. Transcription from an active promoter results in the synthesis of the β-galactosidase that hydrolyses lactose or chromogenic substrates such as o-nitrophenyl-b-D-galactopyranoside (ONPG). Hydrolysis of ONPG results in the formation of o-nitrophenol, which absorps light with a wavelength of 420 nm. The amount of o-nitrophenol that is formed within a certain time is directly proportional to the activity of a promoter. Each condition should be independently measured at least two times.

**Determination of β-galactosidase activity in crude extracts from *B. subtilis***

##### The Material

* LD-Mix (Lysozyme / DNase I)
* Z buffer
* ONPG
* PNPX
* 1 M Na2CO3 solution
* β-Mercaptoethanol

##### The Procedure

1. Propagate a single colony over a whole SP agar plate and incubate the plate over the day at 37°C. You could also propagate a single colony on an SP agar plate in the evening and use the plate at the evening next day to inoculate a pre-culture.
2. Take a single colony of the  *B. subtilis* strain of interest and inoculate 2 - 5 ml C minimal medium supplemented with appropriate antibiotics and effectors (carbon and nitrogen sources). Incubate the pre-cultures over night at 28°C with agitation (220 rpm).
3. Inoculate 10 ml C minimal medium (plus effectors, carbon and nitrogen sources) supplemented in a 100 ml shake flask to an OD*600* of about 0.1 and grow the cultures to an O*D600* of 0.5 - 0.8 (3 – 5 h) at the desired temperature (usually at 37°C for *B. subtilis*).
4. Collect the cells from 1.5 - 2.0 ml of the cultures by centrifugation for 5 min at 13,000 rpm and 4°C. Discard the supernatants and store the cell pellets for further treatment at -20°C.
5. Re-suspend the cells in 400 ml Z buffer (with β-ME and 20 ml LD-Mix in 4 ml Z buffer). Incubate the suspension for 10 min at 37°C to lyse the cells. Keep a minimum of 20 ml Z buffer/LD-Mix for the Bradford assay.
6. Remove cell debris from the soluble fraction by centrifugation for 2 min at 13,000 rpm and 4°C. Transfer the supernatants into new Eppendorf reaction tubes.
7. Add 100 ml of the cell free crude extract to 700 ml Z buffer (with β-Me) and pre-incubate the mixture for 5 min at 28°C. 800 ml Z buffer (with β-ME) will serve as the reference.
8. Start the enzymatic reaction by the addition of 200 ml ONPG to the reaction tubes and note the time.
9. As soon as the reaction mixture turns yellow, stop the reaction by adding 500 ml Na2CO3 and note the time. Now it is time to measure the absorption of the sample at a wavelength of l = 420 nm. The “reference sample” will serve as the reference!
10. The specific β-galactosidase activity in Miller Units/mg protein (MU/mg) can be calculated using the formula below:

    ![](https://static.igem.org/mediawiki/2018/3/3a/T--Goettingen--experiments-galactosidase_activity_formula.png)

    A420: absorption of o-Nitrophenol, Dt: time difference between start and stop of the enzymatic reaction in min, V: volume of crude extract used for the reaction (usually 0.1 ml, see step 3), A595 ∙ 1.7: absorption at l = 595 nm obtained by the Bradford assay.

The method to detect the β-galactosidase activity can also be applied to detect the enzymatic activity of the b-xylosidase in cell free crude extract of *B. subtilis* (Lindner et al., 1994). To detect b-xylosidase activity, ONPG has to be replaced by PNPX.

### Determination of alpha-amylase activity

The amyE gene, encoding the a-amylase can be used to integrate promoter-reporter gene fusions into the chromosome of  *B. subtilis* . Integration occurs by double-homologous recombination via the 5’- and 3’- ends that are flanking the promoter-reporter gene fusion in plasmids pAC5, pAC6 and pAC7 (Martin-Verstraete at al., 1994; Stülke et al., 1997; Weinrauch et al., 1991). The integration of the promoter-reporter gene fusion results in the inactivation of the amyE gene and the lack of a-amylase activity. As the functionality of the amyE gene can easily be determined by monitoring the activity of the a-amylase, the lack of this enzymatic activity is a good indication for the correct integration of the promoter-reporter gene fusion. *B. subtilis*  transformants that did not integrate the promoter-reporter gene fusion by double-homologous recombination (unstable integration) or not at all are still capable of producing α-amylase.
For detecting a-amylase activity, the transformants and a positive control (wild type strain 168) have to be streaked on agar plates containing starch. amyE-positive strains, which synthesis α-amylase hydrolyse the starch and the bacteria are surrounded by a halo when the starch has been stained with Lugol solution. amyE-negative strains do not hydrolyse starch and show no halo formation.

![](https://static.igem.org/mediawiki/2018/c/cd/T--Goettingen--methodamyE.png)

##### The Material

* 5x Lugol (Iodine/K Iodide) solution
* Starch medium agar plates

##### The Procedure

1. Take single colonies of the  *B. subtilis* transformants and a positive control, and propagate the cells on a line on top of the starch plate. Incubate the plates over night at 37°C.
2. Dilute the Lugol solution to 1-fold and add 5 ml of the solution on the plate.
3. The amyE positive bacteria form a halo while those with a disrupted amyE gene should not form a halo.

![](https://static.igem.org/mediawiki/2018/6/6b/T--Goettingen--methodstarch.png)

4. Put the plates under the fume hood and let the plates dry.

### *E. coli* XL1 red-aided mutagenesis of plasmid DNA

For the analysis of important residues in a protein of interest (e.g. protein interaction surface, catalytic site) a random mutagenesis approach can be very helpful. One method for randomly inserted mutations in a gene is the error-prone PCR. The *E. coli* XL1-Red-aided DNA mutagenesis is a very good tool for random mutagenesis of genes, which have been already inserted into vector. Nevertheless, for very small genes (less than 100 bp) or if multiple changes are desired the error-prone PCR is recommended.
 The strain XL1-Red is deficient for three of the primary DNA repair pathways. The mutS (error- prone mismatch repair), mutD (deficient in 3´- to 5´-exonuclease of DNA polymerase III) and mutT (unable to hydrolyze 8-oxodGTP) are affected by mutations introduced into the XL1-Red strain. The random mutation rate in this triple mutant strain was measured to be ~ 5,000-fold higher than that of the wild type (Greener & Callahan, 1994; Greener et al., 1997). The mutated DNA repair pathways are especially important during replication. Therefore, it is very important to achieve a certain generation time to allow the emergence of mutations. For high-copy- number plasmids overnight growth (30 generations) results in approximately one base change per 2,000 nucleotides. For a gene with a size of 2 kb every single clone should contain one point mutation and for a gene that is 1 kb half of the clones will harbor a mutation. This should be considered for the experimental setup. Low-copy vectors or vectors containing very small inserts should be propagated for additional generations. The culture may be diluted for continued growth until stationary phase is reached. Mutations achieved by this method were transitions, transversions, and 1-bp insertions.
 The  *E. coli*  XL1-Red strain grows extremely slowly in rich media (e.g. LB medium), having a doubling time of ~ 90 – 120 minutes. Additionally, the XL1-Red mutator strain should not be propagated on plates for prolonged periods of time. The rapid mutation rate affects the chromosome, and after prolonged growth, the subsequent colonies are probably not genetically identical to the original strain.
 The  *E. coli*  XL1-Red-aided DNA mutagenesis method has been successfully applied to isolate mutant variants of the glutamate dehydrogenase RocG and the glutamine synthetase GlnA in *B. subtilis*  (Wray et al., 2006; Gunka et al., 2010).

##### The Material

* Competent *E. coli*  XL1-Red cells
* Liquid LB medium
* LB medium agar plates with the appropriate antibiotics
* NucleoSpin® plasmid kit, Macherey & Nagel

##### The Procedure

1. Use some material of a cryo culture of the  *E. coli* strain XL1-Red to inoculate 4 ml LB liquid medium and grow the culture overnight at 37°C with agitation.
2. Competent cells of the strain XL1-Red can be prepared according to CaCl2-method and transformed with the respective plasmid to the standard protocol.
3. Plate the cells on an appropriate amount of plates to obtain enough clones. The amount of clones that are needed depends on the size of the gene. We have plated 40 LB plates resulting in approximately 80 colonies per plate for the mutagenesis of an insert with a size of about 1.3 kb (rocG gene) into a vector with a size of ca. 6.7 kb (the plasmid pBQ200). Briefly, we used 3,200 clones for the experiment. For a gene that is about 1.3 kb, half of the clones (1,600) should have a mutation in the insert. Statistically, the resulting plasmid pool should contain a mutant for every base in the gene (Gunka et al., 2010).
4. Re-suspend the colonies from each plate in 1 ml of LB medium, and 100 μl of each suspension can be used to inoculate 100 ml shake flasks containing 10 ml of LB medium supplemented with the appropriate antibiotic.
5. Grow the cultures for 48 h at 37°C to allow the emergence of mutations. If you use a low-copy plasmid or if you mutate a very small gene (less than 1 kb) the culture can be diluted for continued growth.
6. Isolate the plasmid DNA from each culture individually (harvest 3 ml of the culture) by using the NucleoSpin® plasmid kit (Macherey & Nagel low-copy plasmid protocol).
7. An indicator strain (*E. coli*  or  *B. subtilis*) can be transformed with the plasmid pools and the transformants can be screened for the desired phenotype. We have transformed a *B. subtilis*  indicator strain with 40 plasmid pools. Approximately 40,000 clones were screened for a certain phenotype and we have isolated more than 20 interesting plasmids. In this experiment all plasmids contained a single point mutation in the insert (Gunka et al., 2010).

### Fluorescence microscopy

Fluorescence microscopy is a versatile method to investigate protein localization and/or the activity of a promoter. Here we describe a method that is suitable to monitor the activity of a promoter-fluorophore gene fusion in single cells of *B. subtilis* .

##### The Material

* 1x PBS buffer, pH 7.5
* Microscope, microscopy slides and cover slides
* 1% Agarose in water

##### The Procedure

1. Use a single colony of *B. subtilis* to inoculate 4 ml of LB medium supplemented with the appropriate antibiotics. Incubate the culture over night at 37°C with agitation.
2. Next morning inoculate 10 ml LB liquid medium supplemented in a shake flask to an approximate OD600 of 0.05. Incubate the shake flask at 37°C and 200 rpm in the dark and collect the cells at the growth phase of interest.
3. To harvest the cells, transfer 1 ml into a 1.5 ml lightproof (brown) Eppendorf reaction tube and centrifuge the tube for 1 min at 13,000 rpm. Discard the supernatant and Re- suspend the cells in 1x PBS buffer (in a volume (µ{l) = OD600 x (1/10) (µl)). Keep the cell suspension on ice until microscopy.
4. Preparation of the agarose slide for microscopy: boil the agarose, put some agarose on the slide and place the cover slide on top of the agarose; remove the cover slide from the rigid agarose and pipet your cells on top of the agarose; finally, cover the cells with the cover slide.
5. Fluorescence images will be obtained with an Axioskop 40 FL fluorescence microscope, equipped with digital camera AxioCam MRm. The AxioVision Rel 4.8.2 software will be used for image processing (Carl Zeiss, Göttingen, Germany). We will use objective of the Neofluar series is used to produce a 100 X primary magnification. The applied filter sets is the EGFP HC-Filterset (BP: 472/30, FT 495, LP 520/35; AHF Analysentechnik, Tübingen, Germany) for GFP detection. Images can be taken with different exposure times (e.g. 2 sec)

### Competition experiment

Intraspecies competition experiments are an appropriate tool to monitor how mutations of certain genes can affect the fitness of the respective bacterial population under certain conditions. Co-cultivation of two strains which differ in a single locus on the chromosome and which express the fluorophores YFP and CFP is an illustrative way to visualize the competition between those strains.

![]( https://static.igem.org/mediawiki/2018/3/32/T--Goettingen--methodcompetition.png)

##### The Material

* LB medium
* SP agar plates
* Minimal medium (or the desired medium)
* Glycerol (50%)
* NaCl (0.9%)

##### The Procedure

1. Use a fresh colony of the  *B. subtilis* strains of interest, one strain should express YFP and the other strain CFP, to inoculate 4 ml LB medium with the appropriate antibiotics. Incubate the cultures overnight at 28°C and 220 rpm.
2. Prepare cryo cultures with an OD600 of 1.0 of the overnight cultures.
3. Prepare three pre-cultures of the cfp- and yfp-labeled strains. Inoculate 4 ml LB medium containing the appropriate antibiotics with 1 µl of the cryo culture which have an OD600  of 1.0. Incubate the cultures overnight at 28°C and 220 rpm.
4. The next morning, measure the OD600 of the pre-cultures of each strain and use those cultures, which have a similar OD600 between 1.0 and 1.5.
5. In 100 ml shake flasks inoculate 20 ml of minimal medium (or the medium in which you will perform the competition experiment) to an OD600 of 0.05 with each of the two strains you want to analyze. This step ensures that your strains are mixed in a 1:1 ratio at the beginning of the experiment.
6. Harvest directly 10 ml of the culture (point of time t0) in a 15 ml falcon tube by centrifugation for 10 min at 4,000 g.
7. Re-suspend the pellet in 500 µl LB medium and transfer the cells to a sterile 1.5 ml Eppendorf cup. Prepare a cryo culture by adding 500 µl of 50% glycerol. Store the cryo culture at -80°C.
8. Incubate the rest of the culture at 37°C and 220 rpm for 24 h in the dark.
9. After 7 h (time t7) and after 24 hrs (time t24) take additional samples from the growing culture. Measure the OD600 and prepare with appropriate amounts of cells cryo cultures of OD600 1.0. Store them at -80°C.
10. After all the samples are taken, thaw the cryo cultures on ice and dilute the cells in 0.9% saline up to a dilution of 10-3.
11. For counting of the survivors, plate 100 µl of the 10-3 dilution on SP plates with a sterile glass pipette and incubate the plates over night at 37°C in the dark.
12. For illustrative figures, you can additionally spot 10 µl of the 10-4 dilution of the samples from the different time points next to each other on a SP plate, which you incubate over night at 37°C in the dark.
13. The next day, you can count the grown colonies (from step 11.) under the stereo fluorescence microscope. Using the YFP and CFP filter set, the survivors of the yfp- labeled and cfp-labeled strain, respectively, can be made visible and you can exactly determine the outcome of the competition experiment. (Dividing the plate with a black pen into four parts and labelling the already counted colonies makes counting easier!) With respect to the total number of colonies on the plate, calculate the percentage of „blue“ and „yellow“ colonies.
14. To make the clonal composition of the cell population visible at a glance, analyze the spotted samples. Use the cold light source of the stereo fluorescence microscope to bring the spot into focus. Switch the filter to the CFP-filter and take a picture. Do the same for the YFP-filter without moving the plate.
15. With Adobe Photoshop construct merged pictures of the pictures taken from the different spots.

![](https://static.igem.org/mediawiki/2018/4/46/T--Goettingen--methodcompetition2.png )

**To exclude that expression of the fluorophore-encoding genes influences the fitness of the respective strain:**

1. Perform the experiment with inversely labeled strains (the result should be comparable to the actual experiment)
2. Perform the experiment with isogenic strains labeled with cfp and yfp (no negative effect should be detectable)

## Recipes for media, buffers & solutions

### Complex media

##### LB medium

* 10 g Tryptone
* 5 g Yeast extract
* 10 g NaCl
* Ad to 1,000 mL with deionized H2O

##### SP medium

* 8 g Nutrient broth
* 0.25 g MgSO4·7 H2O
* 1 g KCl
* Ad to 1,000 mL with deionized H2O
* *After autoclaving do not forget to add:*
* 1 mL 0.5 M CaCl2
* 1 mL 10 mM MnCl2
* 2 mL Iron Ferric Ammonium Citrate, 2.2 mg/mL

##### Starch medium

* 7.5 g Nutrient broth
* 5 g Starch
* Ad to 1,000 mL with deionized H2O

##### SOB-Mg medium

* 20 g Tryptone
* 5 g Yeast extract
* 0.58 g NaCl
* 0.186 g KCl
* Ad to 1,000 mL with deionized H2O

##### SOB medium

* SOB-Mg medium
* 10 mM MgCl2
* 10 mM MgSO4

### Minimal media

##### CS-glucose medium

* 10 mL 5x C salts

+ 20 g KH2PO4
+ 80 g K2HPO4·3 H2O
+ 16.5 g (NH4)2SO4

* 500 µL Tryptophane
* 500 µL III' salts

+ 0.232 g MnSO4·4 H2O
+ 12.3 g MgSO4·7 H2O
+ Ad to 1,000 mL with deionized H2O

* 500 µL Glucose 50%
* 500 µL Iron Ferric Ammonium Citrate, 2.2 mg/mL
* 1 mL Na-Succinate
* Ad to 50 mL with deionized H2O

##### 10x MN medium

* 136 g K2HPO4·3 H2O
* 60 g KH2PO4
* 10 g sodium citrate · 2 H2O
* Ad to 1,000 mL with deionized H2O

##### MNGE medium

* 1 mL 10x MN medium
* 400 µL Glucose (50%)
* 50 µL Potassium glutamate 40%)
* 50 µL Iron Ferric Ammonium Citrate, 2.2 mg/mL
* 100 µL Tryptophane
* 30 µL 1 M MgSO4
* 8.37 mL deionized H2O
* +/- 100 µL CAA (10%)

### Buffers

##### 50x TAE buffer

* 242 g Tris-base
* 57.1 mL Acetic acid (100%)
* 100 mL 0.5 M EDTA, pH 8.0
* Ad to 1,000 mL with deionized H2O

##### 10x CCR/MMR buffer

* 200 mM Tris-HCl, pH 8.5
* 30 mM MgCl2
* 0.5 M KCl
* 5 mM NAD+
* 0.4 mg/mL BSA

##### Buffer W

* 12.114 g Tris-base
* 8.77 g NaCl
* 0.3722 g Na2EDTA · 2 H2O
* *Adjust the pH with HCl to 8.0*
* Ad to 1,000 mL with deionized H2O

##### Buffer E

* 0.027 g D-Desthiobiotin (25 mM)
* Add 50 mL buffer W

##### Fixation solution

* 10% Acetic acid
* 45% Methanol (toxic!)

##### Staining solution

* 0.5% Coomassie Brillant blue
* 10% Acetic acid
* 45% Methanol (toxic!)

##### De-staining solution

* 5% Acetic acid
* 20% Ethanol

##### 10x PAGE buffer

* 144 g L-Glycine
* 30 g Tris-base
* 10 g SDS
* Ad to 1,000 mL with deionized H2O

### Solutions

##### IPTG

* 2.38 g IPTG
* Ad to 10 mL with deionized H2O
* Sterilization by filtration, store at -20°C in aliquots.

##### 5y DNA loading dye

* 5 mL Glycerol (100%)
* 4.5 mL deionized H2O
* 0.2 mL 50x TAE
* 0.01 g Bromphenol blue
* 0.01 g Xylene cyanol

##### 6x SDS loading dye

* 2.5 mL Tris-HCl, pH 6.8
* 600 µL β-Mercaptoethanol
* 1.2 g SDS
* 7.5 mL Glycerol
* 150 µL Bromphenol blue (2% in EtOH)

##### Expression mix

* 500 µL Yeast extract (5%)
* 250 µL CAA (10%)
* 50 µL Tryptophane (5 mg/mL)
* 250 µL sterile H2O
* **Suitable for 10 samples**

##### 5x Lugol solution

* 100 g K-iodide
* 50 g iodine
* Ad to 1,000 mL with deionized H2O

##### LD-Mix

* 100 mg Lysozyme
* 10 mg DNase I
* Ad to 10 mL with deionized H2O
* Store 500 µL aliquots at -20°C

##### Z buffer

* 60 mM Na2HPO4
* 40 mM NaH2PO4
* 1 mM KCl
* 1 mM MgSO4
* 50 mM β-Mercaptoethanol (toxic!)

### Chromogenic substrates

##### X-Gal

* 80 mg 5-Brom-4-chor-3-indoxyl-β-D-galactopyranosid
* Add 1 mL DMF(N,N-Dimethylformamide) or DMSO

##### ONPG

* 4 mg *o*-Nitrophenyl-β-D-Galactopyranoside (ONPG)
* Add 1 mL Z buffer without β-Mercaptoethanol

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Goettingen/Results

⇧

Team Göttingen
 iGEM 2018

![](https://static.igem.org/mediawiki/2018/d/d4/T--Goettingen--maskotchen.png)

Glyphosate on my plate?

![](https://static.igem.org/mediawiki/2018/4/44/T--Goettingen--Main-Page-iGEM-Logo_tinted.png)

[Home](https://2018.igem.org/Team%3AGoettingen)
[News](https://2018.igem.org/Team%3AGoettingen/News)
Team

[Team Members](https://2018.igem.org/Team%3AGoettingen/Team)
[Collaborations](https://2018.igem.org/Team%3AGoettingen/Collaborations)

Project

[Background](https://2018.igem.org/Team%3AGoettingen/Background)
[Description](https://2018.igem.org/Team%3AGoettingen/Description)
[Design](https://2018.igem.org/Team%3AGoettingen/Design)
[Experiments](https://2018.igem.org/Team%3AGoettingen/Experiments)
[Notebook](https://2018.igem.org/Team%3AGoettingen/Notebook)
[InterLab](https://2018.igem.org/Team%3AGoettingen/InterLab)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)
[Results](https://2018.igem.org/Team%3AGoettingen/Results)
[Demonstrate](https://2018.igem.org/Team%3AGoettingen/Demonstrate)
[Improve](https://2018.igem.org/Team%3AGoettingen/Improve)
[Attributions](https://2018.igem.org/Team%3AGoettingen/Attributions)

Parts

[Parts Overview](https://2018.igem.org/Team%3AGoettingen/Parts)
[Basic Parts](https://2018.igem.org/Team%3AGoettingen/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AGoettingen/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AGoettingen/Part_Collection)

[Safety](https://2018.igem.org/Team%3AGoettingen/Safety)
Human Practices

[Human Practices](https://2018.igem.org/Team%3AGoettingen/Human_Practices)
[Survey](https://2018.igem.org/Team%3AGoettingen/Human_Practices/Survey)
[Education & Engagement](https://2018.igem.org/Team%3AGoettingen/Public_Engagement)

Awards

[Applied Design](https://2018.igem.org/Team%3AGoettingen/Applied_Design)
[Hardware](https://2018.igem.org/Team%3AGoettingen/Hardware)
[Measurement](https://2018.igem.org/Team%3AGoettingen/Measurement)
[Model](https://2018.igem.org/Team%3AGoettingen/Model)

[Judging Form](https://igem.org/2018_Judging_Form?id=2586)

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≡

Contact:

|  |  |
| --- | --- |
| E-mail: | igem2018@gwdg.de |
| Adress: | c/o iGEM 2018 |
|  | Department of General Microbiology |
|  | Grisebachstraße 8 |
|  | 37077 Göttingen |
|  | Germany |

We are especially grateful to our numerous sponsors on [gofundme,](https://de.gofundme.com/glyphosate-on-my-plate) who supported our project and made it possible.
 Every public donor, who helped us to achieve our goals got a special place on our bacterial wall of fame and a handdrawn microbe to show our appreciation. A link to that wall can be found here:

[Bacterial Wall of Fame](https://2018.igem.org/Team%3AGoettingen/WallOfFame)

![Sartorius-Sponsor](https://static.igem.org/mediawiki/2018/d/db/T--Goettingen--Main-Page-sartorius.png)
![Uni-Goettingen-Sponsor](https://static.igem.org/mediawiki/2018/f/f6/T--Goettingen--Main-Page-goettingen.png)
![GZMB-Sponsor](https://static.igem.org/mediawiki/2018/8/8c/T--Goettingen--Main-Page-gzmb.png)

## Results

1. [Identification of glyphosate uptake systems](#r_Uptake)
2. [Identification of glyphosate-regulated promoters](#r_Identification)
3. [A reporter-based glyphosate detection system](#r_reporterDetector)
4. [Competition assay for glyphosate detection](#r_Competition)
5. [Engineering bacteria to disarm glyphosate](#r_Disarm)
6. [Characterization of EPSP synthases](#r_EPSP)
7. [References](#r_References)

![](https://static.igem.org/mediawiki/2018/c/c9/T--Goettingen--treppe.png)

## 1. Identification of glyphosate uptake systems

#### 1.1. Interaction between glyphosate and *Bacillus subtilis*

To create a proper detection system using the Gram-positive model bacterium *Bacillus subtilis*, **we first had to evaluate how this organism behaves in the presence of glyphosate**. Previously, it was shown that glyphosate negatively affects growth of *B. subtilis* due to the inhibition of the EPSP synthase AroE (Figure 1A) (1). Moreover, it has been demonstrated that 1.8 mM of glyphosate was required to inhibit the growth rate by 50%. To re-evaluate the effect of glyphosate on growth of our *B.  subtilis* laboratory strains 168 and SP1, we performed growth experiments in CS-Glc minimal medium that was supplemented with increasing amounts of glyphosate (Figure 1B). CS-Glc medium contains glucose and succinate as carbon sources and ammonium as the nitrogen source (see [Notebook](https://2018.igem.org/Team%3AGoettingen/Notebook)). While the *B. subtilis* strain 168 is auxotrophic for tryptophan due to a mutation in the *trpC* gene, the strain SP1 can grow in the absence of exogenous tryptophan (2).

![](https://static.igem.org/mediawiki/2018/c/c8/T--goettingen--GS_EPSP_interaction.jpg)

Figure 1. (A) Glyphosate inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which converts phosphoenolpyruvate (PEP) and 3-P-shikimate into EPSP and P in *B. subtilis*. EPSP is a precursor for the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophane (Trp). (B) How does glyphosate affect growth of our *B. subtilis* wild type strains?

As shown in Figures 2A and 2B, at a glyphosate concentration of about 1 mM the growth rate of both strains was reduced by 50% and the bacteria were not able to grow at glyphosate concentrations higher than 3 mM. In contrast to a previous study (1), we have observed that 44% fewer glyphosate is required to reduce the growth rate of *B. subtilis* by 50%. This discrepancy might be due to differences in the genetic makeup of the *B. subtilis* strains, in the medium composition, in the purity of glyphosate or due to the different cultivation conditions. However, **glyphosate negatively affects growth of *B. subtilis*** in CS-Glc minimal medium.

![](https://static.igem.org/mediawiki/2018/8/86/T--goettingen--168_Sp1_GS_small.jpg)

Figure 2. (A) Growth of the *B. subtilis* wild type strain 168 with increasing amounts of glyphosate (GS). (B) Growth of the *B. subtilis* wild type strain SP1 with increasing amounts of GS. The figure inlays show the relationship between the growth rate (μ) and the GS concentration. The bacteria were cultivated at 37°C in CS-Glc minimal medium.

#### 1.2. Genomic adaptation of *Bacillus subtilis* to glyphosate

Previously, it has been reported that the growth rate of a *B. subtilis* culture containing 2.5 mM glyphosate strongly increases after 16 h of incubation (1). It has been suggested that this phenomenon might be either due to a physiological adaptation of the bacteria to glyphosate or due to the emergence of glyphosate-resistant mutants. To assess whether *B. subtilis* is able to adapt to toxic levels of glyphosate at the genome level, we propagated the wild type strain 168 on CS-Glc agar plates supplemented with increasing amounts of the herbicide (0, 10, 20 30 and 40 mM). After incubation for 24 h at 37°C, bacterial growth was only visible on the plates lacking glyphosate. When the plates were further incubated for 48 h, **several glyphosate-resistant mutants appeared on the plates supplemented with 10 mM glyphosate** (Figure 3A). No mutants appeared on the plates with glyphosate concentrations higher than 10 mM. Next, we repeated the plating experiment with a mutant (designated as iGEM1) that was isolated from the 10 mM glyphosate plate. After 48 h of incubation, the glyphosate-resistant strain iGEM1 formed mutants on plates containing 30 mM and 40 mM glyphosate (Fig. 2A). Again we isolated mutants from the 30 mM and the 40 mM glyphosate plates that were designated as iGEM 7 and iGEM13, respectively (Figure 3B). Our attempts to further increase the glyphosate resistance of the strains iGEM7 and iGEM13 failed. To ensure that the acquired mutations in the strains iGEM1, iGEM7 and iGEM13 are stable, the mutants were passaged three times on SP rich medium plates. The glyphosate resistance of the mutants was confirmed by streaking the bacteria on CS-Glc medium supplemented with increasing amounts of glyphosate. As in Figure 3C, all strains that were adapted to glyphosate grew on plates with 10 mM glyphosate. The strains iGEM7 and iGEM13 also formed single colonies on 20 mM glyphosate plates. Moreover, albeit weak, the strain iGEM13 showed even growth with 40 mM glyphosate.

![](https://static.igem.org/mediawiki/2018/6/6f/T--goettingen--Bacillus_GS_adaptation.jpg)

Figure 3. Isolation and characterization of glyphosate (GS)-resistant mutants.
(A) Emergence of GS-resistant *B. subtilis* mutants on CS-Glc minimal medium agar plates that were incubated for 48 h at 37°C. WT, *B. subtilis* wild type strain 168; iGEM1, GS-resistant *B. subtilis* strain that was isolated from plates containing 10 mM GS.
(B) Genealogy of the GS-resistant *B. subtilis* mutants.
(C) Evaluation of growth of the GS-resistant mutants on CS-Glc minimal medium plates supplemented with increasing amounts of GS. The plates were incubated for 48 h at 37°C.

Our growth experiments using CS-Glc liquid medium with increasing amounts of glyphosate also confirmed that the strains iGEM7 and iGEM13 tolerate higher amounts of the herbicide than their progenitor strain iGEM1 (Figure 4; see below). To conclude, ***B. subtilis* rapidly adapts to toxic glyphosate concentrations at the genome level!**

![](https://static.igem.org/mediawiki/2018/a/a2/T--goettingen--iGEM_1_7_13.jpg)

Figure 4. Characterization of glyphosate (GS)-resistant *B. subtilis* mutants. The wild type (WT) strain (A) and its derivatives iGEM1 (B), iGEM7 (C), and iGEM13 (D) were cultivated for 10 h at 37°C in CS-Glc minimal medium supplemented with increasing amounts of GS.

#### 1.3. Identification of mutations by genome and Sanger sequencing

To identify the underlying genomic alterations causing glyphosate resistance the strains iGEM1, iGEM7 and iGEM13, we performed whole-genome sequencing with the help of the [Göttingen Genomics Laboratory](http://appmibio.uni-goettingen.de/index.php?sec=g2l) (Figure 5A). By analysing the genome sequences we found that the bacteria had deleted a 1240 bp-long fragment containing a part of the 5’ ends of the *yhfE* and *gltT* genes and the entire *yhfF* gene (Figure 5B).

![](https://static.igem.org/mediawiki/2018/7/7b/T--goettingen--Genome_sequencing_gltT_mutations.jpg)

Figure 5. The mutations of the glyphosate-resistant mutants and the link between glutamate and glyphosate transport.
(A) The illumina sequencing reads were mapped onto the reference the *B. subtilis* 168 reference genome NC\_000964 from [GenBank](https://www.ncbi.nlm.nih.gov/nuccore/NC_000964) (3) using the Geneious software package ([Biomatters Ltd.](https://www.geneious.com/about/)) (4).
(B) The mutations in the glyphosate (GS)-resistant *B. subtilis* mutants identified by genome sequencing.
(C) Effect of the identified mutations on the glutamate transporters GltT and GltP in the strains iGEM1, 7 and 13. Membrane topologies of the transporters were illustrated using the web-based protein topology tool [Protter](http://wlab.ethz.ch/protter/start/) (5).
(D) Comparison of glutamate and glyphosate.

The 1240 bp-long deletion is also present in the strains iGEM7 and iGEM13 because they were derived from the strain iGEM1 (Figure 5B). In addition to the deletion affecting the *yhfE*, *yhfF* and *gltT* genes, the strains iGEM7 and iGEM13 had replaced a single nucleotide and deleted 11 nucleotides, respectively, in the *gltP* gene. The *yhfE* gene encodes a putative aminopeptidase and the function of the *yhfF* gene is unknown ([SubtiWiki](http://www.subtiwiki.uni-goettingen.de/v3/gene/view/A666F376947A675E04550338C3177FF8AFF44DF4); 6). The *gltT* and *gltP* genes code for the high-affinity sodium-coupled glutamate/aspartate symporter GltT and for the low-affinity proton/glutamate symporter GltP, respectively (7,8). The fact that all strains had acquired mutations in genes encoding transporters that are involved in glutamate and aspartate uptake suggests that the proteins also transport glyphosate (Figure 5B). The 1240 bp- and 11 bp-long deletions in the *gltT* and *gltP* genes truncate the encoded proteins after 282 and 214 amino acids, respectively (Figure 5C). The G232A mutation in the *gltP* gene results in a replacement of a glutamate residue by a lysine residue at position 78, which is adjacent to the second transmembrane helix of the GltP transporter (Figure 5C). While the deletions certainly destroy the amino acid transporters, the E78K replacement probably reduces the transport of glyphosate *via* GltP. This idea is in line with the observation that the strain iGEM7 carrying the G232A mutation in the *gltP* gene did not grow as good as the strain iGEM13 with the deletion in *gltP* on plates with high amounts of glyphosate (Figure 5C). A structural comparison revealed that glutamate and glyphosate resemble each other because the molecules contain negatively charged groups encompassing positively charged groups (Figure 5D). Thus, both, GltT and GltP could be involved in glyphosate uptake. To conclude, ***B. subtilis* rapidly adapts to toxic glyphosate concentrations by inactivating the *gltT* and *gltP* genes!**

#### 1.4. Glyphosate-resistant mutants preferentially inactivate the *gltT* gene

Previously, it has been shown that bacteria like *Salmonella typhimurium* can develop glyphosate resistance due to the acquisition of mutations in the promoter region and in the coding region of the *aroA* gene, elevating the EPSP synthase levels and rendering the encoded enzyme insensitive to glyphosate, respectively (9-11). Our genome sequencing analyses of the isolated iGEM1, iGEM7 and iGEM13 strains did not identify mutations that alter the synthesis or the coding sequence of the EPSP synthase. To assess whether *B. subtilis* can develop glyphosate resistance by the acquisition of mutations that alter the target of the herbicide, we isolated 12 additional mutants from CS-Glc plates containing 10 mM glyphosate. DNA sequencing analysis revealed that each strain had mutations within the *gltT* coding sequence that would truncate or lengthen GltT transporter (Figure 6). Thus, **the inactivation of the *gltT* gene is the predominant mutational event allowing *B.  subtilis* to grow in the presence of the weedkiller glyphosate**.

![](https://static.igem.org/mediawiki/2018/3/37/T--goettingen--gltT_genes_mutations_black.jpg)

Figure 6. The mutations in the *gltT* gene and the consequences for the encoded transporter GltT in the *B. subtilis* mutants that were isolated from CS-Glc medium agar plates containing 10 mM glyphosate.

#### 1.5. Characterization of *gltT*, *gltP* and *gltT* *gltP* mutant strains

Next, we tested whether the clean deletion of either the *gltT* gene or the *gltT* *gltP* genes is indeed sufficient to confer high-level resistance of *B. subtilis* to glyphosate. For this purpose, we constructed the mutant strains BP233 (*gltT*), BP234 (*gltP*) and BP235 (*gltT* *gltP*). To assess the glyphosate resistance of the *gltT*, *gltP* and *gltT* *gltP* mutants, we cultivated the bacteria in CS-Glc minimal medium supplemented with increasing amounts of glyphosate. As shown in Figure 7A and 7C, growth of the wild type and the *gltP* mutant strains, respectively, was inhibited by 5 mM glyphosate. This observation indicates that **GltP is a low-affinity glyphosate transporter** and that the cell still contains a transport system for the herbicide. **The transporter with a high-affinity for glyphosate seems to be in fact GltT** because the deletion of the *gltT* gene conferred high-level resistance to the herbicide (Figure 7B). Moreover, the deletion of the *gltP* gene in the background of a *gltT* mutant strain further enhanced glyphosate tolerance and the growth rates of the strains BP233 (*gltT*) and BP235 (*gltT* *gltP*) were reduced by 50% at herbicide concentrations of 6.1 mM and 8.0 mM, respectively (Figure 7B and 7D). Thus, in comparison to the wild type strain, 6- to 8-fold higher glyphosate concentrations are needed to reduce the growth rate of the strains BP233 (*gltT*) and BP235 (*gltT* *gltP*) by 50% (Figure 8).

![](https://static.igem.org/mediawiki/2018/b/b2/T--goettingen--168_Sp1_GS_liquid_small_neu.jpg)

Figure 7. Inactivation of the glutamate transporters confers high-level resistance to glyphosate (GS).
(A) Growth of the *B. subtilis* wild type (WT) strain 168 in CS-Glc minimal medium supplemented with increasing amounts of GS.
(B) Growth of the Δ*gltT* mutant strain BP233 in CS-Glc minimal medium supplemented with increasing amounts of GS. The figure inlay shows the relationship between the growth rate (µ) and the GS concentration.
(C) Growth of the Δ*gltP* mutant strain BP234 in CS-Glc minimal medium supplemented with increasing amounts of GS.
(D) Growth of the Δ*gltT* Δ*gltP* mutant strain BP235 in CS-Glc minimal medium supplemented with increasing amounts of GS. The figure inlay shows the relationship between the growth rate (µ) and the GS concentration.

![](https://static.igem.org/mediawiki/2018/e/e4/T--goettingen--iC50.jpg)

Figure 8. The relationship between the growth rate (µ) and the glyphosate (GS) concentration for the *B. subtilis* wild type (WT) strain 168,
the Δ*gltT* mutant strain BP233 and the the Δ*gltT* Δ*gltP* mutant strain BP235 in CS-Glc minimal medium supplemented with increasing amounts of GS.

We also tested whether the glyphosate that is present in commercially available Roundup® is toxic for *B. subtilis*. Roundup® was ordered from the German company
[Westfalia](https://www.westfalia.de/shops/garten/pflanzenschutz_und_duenger/unkrautbekaempfung/pflanzenschutzmittel/1228751-unkrautfrei_alphee_1_liter_die_nr_1_gegen_unkraut.htm). 1 l of Roundup® Alphee contains 9.4 g of the isopropylamine salt of glyphosate (which corresponds to 7.2 g/l pure glyphosate), 5 g surfactant (undefined) and water. To test the effect of Roundup® Alphee on growth of the bacteria, we propagated the *B. subtilis* strains 168 (wild type), BP233 (Δ*gltT*), BP234 (Δ*gltP*) and BP235 (Δ*gltT* Δ*gltP*) on CS-Glc minimal medium agar plates that were supplemented with glyphosate (control) and with equimolar amounts of glyphosate present in Roundup (test conditions). As shown in Figures 9A and 9B, pure glyphosate and **Roundup® Alphee killed the *B. subtilis* wild type strain** and the strain lacking the low-affinity glutamate transporter GltP already at a concentration of 2 mM. By contrast, all strains lacking the high-affinity transporter GltT grew in the presence of 2 - 6 mM glyphosate that is present in Roundup. Thus, **the unknown surfactant that is present in the commercially available Roundup® solution does not seem to affect growth of *B. subtilis***. Moreover, it is worth to mention that **the glyphosate present in the Roundup® solution is rather cheap: 7.2 g of glyphosate would make more than 3 k€ @ [Merck](https://www.sigmaaldrich.com/catalog/product/sial/89432?lang=de&region=DE)**!

![](https://static.igem.org/mediawiki/2018/d/df/T--goettingen--Roundup_gltT_gltP.jpg)

Figure 9. A. Growth of the strains 168 (wild type), BP233 (Δ*gltT*), BP234 (Δ*gltP*) and BP235 (Δ*gltT* Δ*gltP*) on CS-Glc minimal medium in the presence of different amounts of glyphosate (GS). B. Roundup® Alphee purchased from [Westfalia](https://www.westfalia.de/shops/garten/pflanzenschutz_und_duenger/unkrautbekaempfung/pflanzenschutzmittel/1228751-unkrautfrei_alphee_1_liter_die_nr_1_gegen_unkraut.htm). C. Growth of the strains 168, BP233, BP234 and BP235 on CS-Glc minimal medium in the presence of different amounts of Roundup® Alphee.

#### 1.6. Complementation of the *gltT* mutation

We also performed a complementation experiment to provide further evidence that GltT is the major glyphosate transporter in *B. subtilis*. For this purpose, we fused the artificial *Palf4* promoter (12) together with the *gapA* ribosome-binding site to the *gltT* gene and integrated the construct into the *amyE* locus of the *gltT* mutant (Figure 10A). Next, we propagated the strains BP233 (*gltT*) and BP237 (*gltT* *Palf4-gltT*) together with the wild type strain 168 on agar plates without and with glyphosate (Figure 10B). As expected, all strains grew in the absence of glyphosate. By contrast, only the *gltT* mutant strain BP233 grew with glyphosate. Thus, like the wild type also the complementation strain BP237 take up glyphosate *via* GltT. To conclude, under the tested growth conditions **the high-affinity glutamate transporter GltT is the major entryway of glyphosate into *B. subtilis*!**.

![](https://static.igem.org/mediawiki/2018/6/69/T--goettingen--complementation_icon_neu_corrected.jpg)

Figure 10. Complementation of the Δ*gltT* mutant strain.
(A) The artificial *Palf4* promoter (12) and the ribosome-binding site (RBS) of the *B. subtilis gapA* gene were fused by PCR to the *gltT* gene.
(B) Growth of the *B. subtilis* wild type 168 (WT), the Δ*gltT* mutant BP233 and the Δ*gltT Palf4-gltT* complementation strain BP237 on CS-Glc minimal medium agar plates without glyphosate (0 mM GS) and with glyphosate (10 mM GS). The plates were incubated for 24 h at 37°C.

#### 1.7. GltT is also involved in the uptake of the herbicide glufosinate

Our structural comparison of glutamate and glyphosate revealed that the two molecules resemble each other because they contain negatively charged groups encompassing positively charged groups (see Figure 5D). However, the structural similarity of glutamate and glyphosate is not very high. During a scientific discussion with our collaborator  **Dr. Till Ischebeck** from the [Department of Plant Biochemistry](https://www.uni-goettingen.de/de/533683.html) at the University of Göttingen, we came up with the idea that GltT may also transport the herbicide glufosinate, which is structurally more similar to glutamate than glyphosate and inhibits the glutamine synthetase (Figure 11A) (13). Glufosinate (phosphinothricin) is a naturally occurring broad-spectrum systemic herbicide produced by several species of *Streptomyces* soil bacteria. Plants may also metabolize
[bialaphos](https://en.wikipedia.org/wiki/Bialaphos) (L-Alanyl-L-alanyl-phosphinothricin), another naturally occurring herbicide, directly into glufosinate (13). The compound irreversibly inhibits glutamine synthetase, an enzyme necessary for the *de novo* synthesis of glutamine from ammonia and glutamate, giving it antibacterial, antifungal and herbicidal properties. Application of glufosinate to plants leads to reduced glutamine and elevated ammonia levels in tissues, halting photosynthesis, resulting in plant death. In the 1960s and early 1970s, scientists at University of Tübingen and at the [Meiji Seika Kaisha Company](https://www.meiji.com/global/about-us/corporate-profile/meiji-seika-pharma/) independently discovered that species of *Streptomyces* bacteria produce a tripeptide they called bialaphos that inhibits bacteria; it consists of two alanine residues and a unique amino acid that is an analog of glutamate that they named "phosphinothricin".
**The inactivation of the *gltT* gene indeed allowed *B. subtilis* to tolerate glufosinate (Figures 11B and 11C)!** Moreover, the observation that the expression of two copies of the *gltT* gene in strain BP236 (*amyE::Palf4-gltT*) increased the sensitivity of the bacteria toward glufosinate further supports the idea that GltT is involved in glufosinate uptake by *B. subtilis* (Figures 11B and 11C). In contrast to glyphosate, however, the simultaneous deletion of the *gltT* and *gltP* genes did not increase glufosinate tolerance of *B. subtilis* (Figures 11B and 11C). To conclude, under the tested growth conditions **the high-affinity glutamate transporter GltT is the major entryway of glyphosate into *B. subtilis* and GltT is also involved in Glufosinate uptake!**

![](https://static.igem.org/mediawiki/2018/a/a1/T--goettingen--Glufosinate_icon_neu.jpg)

Figure 11. Glufosinate is also taken up by *B. subtilis* *via* GltT.
(A) Comparison of glutamate and glufosinate.
(B) Disk diffusion assay to test whether glufosinate is transported *via* GltT and/or GltP. The strains 168 (wild type), BP233 (Δ*gltT*), BP234 (Δ*gltP*, BP235 (Δ*gltT* Δ*gltP*), BP237 (Δ*gltT* *amyE::Palf4-gltT*) and BP236 (*amyE::Palf4-gltT*) were cultivated in LB medium, washed once in 1 X C-salts and propagated (100 µl cell suspension, OD600 ≈ 2) on CS-Glc minimal medium agar plates. A filter disk containing 5 µl of a 0.5 M glufosinate solution was placed in the center of the plates, which were incubated for 2 days at 37°C. (C) Evaluation of the disk diffusion assay shown in (B). (D) Killing two birds with one stone: the Δ*gltT* mutation confers resistance to glyphosate and glufosinate!

#### 1.8. A novel nechanism conferring resistance to glyphosate

In the past years, the underlying molecular mechanisms conferring resistance to glyphosate have been intensively studied in bacteria and plants. **In many cases glyphosate resistance is directly linked to the target of the herbicide**. For instance, bacteria like *S. aureus* are *a priori* resistant to glyphosate because this organism synthesizes an insensitive EPSP synthase (14-17). Moreover, increased cellular levels of the EPSP synthase, which can be achieved either by overexpression of the coding gene or by gene amplification, can confer resistance to glyphosate (Figure 12) (9, 18-22). Due to the increased cellular levels of the EPSP synthase, the glyphosate is probably titrated away and sufficient amounts of the precursor for aromatic amino acid biosynthesis can be produced. As mentioned in the design section, several glyphosate-insensitive EPSP synthase mutant variants have been isolated and engineered (11, 23-36). Often, a single amino acid exchange is sufficient to render the EPSP synthase insensitive to glyphosate. However, although glyphosate resistance is frequently linked to the target of the herbicide, resistance against the herbicide may occur by other means. Several studies have demonstrated that glyphosate can be detoxified by covalent modification. For instance, the Gat glyphosate *N*-acetyltransferase from *Bacillus licheniformis*, which was subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide, converts glyphosate to *N*-acetylglyphosate, which is not herbicidal and is not an effective inhibitor of EPSP synthases (Figure 12) (37-40). Moreover, the hygromycin phosphotransferases Hph and GlpA from *E. coli* and *Pseudomonas pseudomallei*, respectively, phosphorylate glyphosate and thus confer tolerance to the herbicide (Figure 12) (41,42). Interestingly, the *gat* gene has also been used as a selection marker for genetic engineering of bacteria (40). The enzymes that covalently modify glyphosate have been successfully introduced into crops to increase herbicide resistance (43).
Recently, it has been also demonstrated that enhanced export of glyphosate can reduce toxicity of the herbicide. For instance, the overexpression of the uncharacterized membrane proteins MFS40 and YhhS from *Aspergillus oryzae* and *E. coli*, respectively, with similarity to the major facilitator secondary transporter superfamily, enhance glyphosate tolerance of *E. coli* (Figure 12) (44,45). It will be interesting to test whether these transporters are suitable for engineering crops to enhance glyphosate tolerance. Proteins of unknown function can also increase glyphosate tolerance. For instance, the overexpression of the *igrA* gene product from the *Pseudomonas* sp. strain PG2982 increases glyphosate tolerance in transgenic rice (46-48). Finally, many bacteria can survive in the presence of glyphosate because they are able to degrade the herbicide (Figure 12) (49-58). To conclude, **several mechanisms of glyphosate resistance have been described over the past years and novel mechanisms allowing survival with the herbicide are certainly identified in the future**.
We have observed that the deletion of the *gltT* gene in *B. subtilis* confers high-level glyphosate resistance to the bacteria. **The *B. subtilis* *gltT* mutant strain could be a suitable host to identify glyphosate uptake systems from plants by expressing cDNA libraries and by screening for transformants that are unable to grow with glyphosate**.

![](https://static.igem.org/mediawiki/2018/9/98/T--goettingen--resistance_mechan_icon.jpg)

Figure 12. Mechanisms conferring resistance to glyphosate.
GltT and GltP, *B. subtilis* glutamate transporters; YhhS and MFS40, major facilitator secondary transporters from *E. coli* and *A. oryzae*, respectively; GAT, *B. licheniformis* glyphosate *N*-acetyltransferase; GlpA, *P. pseudomallei* hygromycin phosphotransferase; PEP, phosphoenolpyruvate; 3PS, 3-phosphoshikimate; EPSP, enolpyruvylshikimate-3-phosphate; EPSPS, enolpyruvylshikimate-3-phosphate synthase. Membrane topologies of the transporters were illustrated using the web-based protein topology tool [Protter](http://wlab.ethz.ch/protter/start/) (5).

## 2. Identification of glyphosate-regulated promoters

#### 2.1. Effect of glyphosate on the *Bacillus subtilis* transcriptome

The identification of genes that are up- or down-regulated in a glyphosate-dependent manner requires the application of "omics" techniques such as proteomics or transcriptomics. RNA-sequencing (RNA-Seq) analysis is a valid methodology to identify genes that are either induced or repressed by chemical substances such as nutrients and herbicides.
To assess the effect of glyphosate on the *B. subtilis* transcriptome, we performed an RNA-Seq analysis with the help of the [Göttingen Genomics Laboratory](http://appmibio.uni-goettingen.de/index.php?sec=g2l). The effect of glyphosate on the *Escherichia coli* transcriptome has been analyzed in the past years (59). By contrast, the effect of the weedkiller on gene expression in *B. subtilis* has remained elusive. To obtain bacterial cell pellets for RNA isolation and subsequent RNA-Seq analysis, we cultivated the *B. subtilis* wild type strain SP1 in CS-Glc minimal medium without glyphosate and supplemented with 0.75 mM glyphosate. We selected a concentration of 0.75 mM because it was previously shown that at a glyphosate concentration of about 1 mM the growth rate of the strain SP1 was reduced by 50% (see Figure 2). By choosing a lower concentration, we wanted to ensure that the bacteria were under stress without affecting growth too severe.

![](https://static.igem.org/mediawiki/2018/2/22/T--Goettingen--RNA_Seq.png)

Figure 13. Effect of glyphosate on the *B. subtilis* transcriptome. (A) The diagram shows the fold change of the *B. subtilis* RNAs during cultivation in presence of glyphosate. The strongest regulated operon is marked with yellow circles. (B) A list of genes whose expression was severely affected by glyphosate. The four most down- and up-regulated genes (marked in (A)).

The RNA-Seq analysis allowed us to identify genes whose expression is altered in a glyphosate-dependent manner. As illustrated in Figure 13A, the expression of only a few genes was affected by glyphosate. A 5-fold increase in gene expression was the highest increase we could observe. By contrast, we observed that the expression of some genes was 11-fold reduced. Nearly all up-regulated genes are involved in sporulation and **the *yciA-yciB-yczL-yciC* operon whose expression is affected by glyphosate is related to zinc deprivation responses** (60). Unexpectedly, we could not detect any effect of glyphosate on the expression of genes encoding proteins involved in shikimate metabolism and aromatic amino acid biosynthesis. This finding could indicate that the inhibition of the EPSP synthase and the changes of the cellular EPSP concentration does not influence the expression of the genes that are involved in aromatic amino acid biosynthesis in *B. subtilis*.
The promoters involved in the regulation of the *yciA-yciB-yczL-yciC* operon were **identified as PyciAB and PyciC (61).**

## 3. A reporter-based glyphosate detection system

#### 3.1 Utilising PyciAB and PyciC to build a reporter-based glyphosate detection system

Our RNA-Seq analysis revealed that the expression of the operon containing the *yciA-yciB-yczL-yciC*genes was severely affected when *B. subtilis* was cultivated in the presence of glyphosate. The promoters involved in the regulation of these genes were identified as PyciAB and PyciC. Based on our RNA-Seq data, we aimed to construct a reporter-based glyphosate detection system. For this purpose, we fused the identified promoters to the *xylR* gene, which is a transcriptional repressor of the *xylA-xylB* and *xynP-xynB* operons in *B. subtilis* (Figure 14A). A *B. subtilis* mutant strain lacking the *xylR* gene was transformed with the plasmid pAC7 containing our promoter-gene constructs. We expect that upon contact of the constructed strains glyphosate, the expression of the *xylR* gene will be lower, which will lead to the expression of the *xylA* reporter gene. The expression of the *xylA* reporter gene can be monitored by performing a β-xylosidase assay using the colourimetric substrate 4-Nitrophenyl-β-D-xylopyranoside (PNPX). The measured β-xylosidase activity can be directly linked to the expression of the *xylR* gene, which controlled by the glyphosate-dependent promotor (Figure 14B). **The β-xylosidase activity should be directly proportional to the glyphosate that is present in the growth medium.**

![](https://static.igem.org/mediawiki/2018/c/c3/T--Goettingen--Reportersystem2.0.png)

Figure 14. (A) Sequence of the promotores PyciAB and PyciC including the ribosome-binding site (RBS) were fused to the *xylR* gene by PCR. (B) Mode of action of the glyphosate detection system.

By performing a β-xylosidase assay we could determine that this system does not react to the presence of glyphosate. This might be due to the fact, that the promotores are not regulated or the regulation is not strong enough.

## 4. Competition assay for glyphosate detection

#### 4.1. Competition assay with fluorophores

Unfortunately, current analytical methods, which are based on enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry (MS) are very expansive, slow, need sophisticated equipment and well-trained technical staff. Therefore, **the development of a rapid and straightforward method to detect glyphosate is very attractive because the intensive use of the herbicide may lead to its accumulation in the soil and food**. The basic idea of the competition assay for the detection of glyphosate is depicted in Figure 15.

![](https://static.igem.org/mediawiki/2018/3/39/T--Goettingen--competitionassay_overview_als_jpg_mithintergrund.png)

**Figure 15.** The basic principle of the competition assay for glyphosate detection. In the absence of glyphosate (no selective pressure), both strains grow with the same rate. When glyphosate is present, the herbicide-resistant strain (expressing an orange fluorophore gene) will outcompete the wild type strain. Thus, the velocity at which the wild type strain is eliminated from the co-culture is indicative for the glyphosate present in the culture or test sample.

Two *B. subtilis* strains that tolerate different amounts of glyphosate are labelled with different fluorophore genes (Figure 16). The fluorescence labelling of the bacteria allows to count the amount of each strain when co-cultivated in the absence and in the presence of selective pressure that is exerted by glyphosate using fluorescence microscopy. In the absence of selective pressure, we expect that both strains will survive the co-cultivation. By contrast, in the presence of glyphosate, the strain that tolerates glyphosate will outcompete the glyphosate-sensitive *B. subtilis* strain. Thus, **the selective pressure (≈ [glyphosate]) correlates with the amount of glyphosate-resistant cells and glyphosate-sensitive cells.**

![](https://static.igem.org/mediawiki/2018/0/0a/T--Goettingen--fluorophore_gene_in_amyE.png)

**Figure 16.**  Integration of the fluorophore genes whose expression are controlled by an artificial σA-dependent promoter (*Plij2*), which is constitutively active. The constructs are be integrated into the *amyE* gene of the *B. subtilis* chromosome in single copy. RBS, ribosome-binding site; ORF, open reading frame.

##### Characterization of the mOrange protein in *B. subtilis*

The fluorophore genes are derived from the iGEM DNA distribution kit. Previous work using the [BBa\_E2050](http://parts.igem.org/Part%3ABBa_E2050) biobrick
was focused on the function of the mOrange protein (an mRFP (DsRed) derivative) in the yeast *S. cerevisiae*. The aim of our experiments was
to integrate the gene encoding the mOrange protein into the genomes of different *B. subtilis* strains. Unfortunately, the plasmid pSB1C3 does not contain an origin of
replication for *B. subtilis*. Therefore, we introduced the fluorophore genes into the *B. subtilis* plasmid pAC7. For this purpose, we amplified the fluorophore genes by PCR, ligated the digested PCR products with the plasmid pAC7 that was cut with the same enzymes and transformed competent cells of the *E. coli* strain DH5α. During PCR, the fluorophore genes were fused to the artificial (*Plij2* promoter, which should be constitutively active (see Figure 16). The promoter was designed *in silico* and contains binding sites for the housekeeping sigma factor A and a perfect RBS for *B. subtilis*. As shown in Figure 17, all strains synthesizing mOrange formed cell pellets with an orange color.

![](https://static.igem.org/mediawiki/2018/archive/0/0c/20181010080841%21T--Goettingen--characterization_pellets.png)

Figure 17. A cell pellet of the *B. subtilis* wild type strain 168 is not orange. The cell pellets of the strains iGEM24, iGEM28 and iGEM36 synthesizing mOrange are slightly colored in orange.

In the next step, the plasmid containing the mOrange gene was used to transform the *B. subtilis* wild type strain 168 and the glyphosate resistant strains BP233 (Δ*gltT*) and BP235 (Δ*gltT* Δ*gltP*). The activity of the mOrange fluorophore protein in the *B. subtilis* wild type strain 168 was further analyzed by fluorescence microscopy. Therefore, the strain was cultivated overnight at 37°C in the darkness. 0.01 ml of the culture were transferred to a microscope slide that was covered with solid 1% agarose dissolved in H2O to inhibit the movement of the cells during long-time exposure. As shown in Figure 18, cells synthesizing the mOrange fluorophore protein were highly fluorescent. Thus, **the mOrange fluorophore protein, which is part of the [BBa\_E2050](http://parts.igem.org/Part%3ABBa_E2050) biobrick can be produced in *B. subtilis***.

![](https://static.igem.org/mediawiki/2018/c/c6/T--Goettingen--Characterization_microscope_hi.png)

Figure 18. The left panel shows cells of the strain iGEM24 (wild type strain producing mOrange) that were visualized using bright field microscopy. Magnification = 40 X; exposure time = 0.85 s; no filter. The right panel shows cells of the same strain visualized by fluorescence microscopy. Magnification = 40 X; exposure time 44.33 s; DAPI filter.

**To create a powerful glyphosate detection system, which is based on intraspecies competition, we need to have strains in hands having similar fitness coefficients**. Strains having a different fitness will not allow to generate a stable co-culture because the strain with the higher fitness will alway outcompete the other strain, even in the absence of a selective pressure. To characterize our strains and to determine their fitness we performed different growth experiments. First, we integrated fluorophore genes into the *amyE* gene of the *B. subtilis* strains 168, (wild type), Δ*gltT* (BP233) and Δ*gltT* Δ*gltP* (BP235). The wild type strain 168 is very sensitive to glyphosate (see Figure 2). By contrast, the Δ*gltT* single mutant and Δ*gltT* Δ*gltP* double mutant lacking the glyphosate transport systems tolerate high amounts of the herbicide (see Figures 7 and 8). We used the *gfp*, *bfp* and *morange* genes encoding the fluorophores GFP, BFP and mOrange, respectively, for strain labeling. To drive the expression of the fluorophore genes, we attached an artificial promoter to the genes during PCR (see Figure 16). The sigma factor A-dependent artificial promoter is constitutively active and contains a ribosome binding site for *B. subtilis*. Next, we cultivated the constructed strains in a multi-well plate reader, to determine their fitness based on the growth rates that are achieved in the medium.

![](https://static.igem.org/mediawiki/2018/a/ae/T--Goettingen--competitionassay_growthderorangenen.png)

**Figure 19.** Growth of the strains iGEM24 (*mOrange*), iGEM28 (Δ*gltT mOrange*) and iGEM36 (Δ*gltT* Δ*gltP mOrange*) in CS-Glc minimal medium. All strains synthesizing mOrange have the same growth rate.

As shown in Figure 19, all strains had similar growth rates in CS-Glc minimal medium. Thus, **the expression of the mOrange gene and the absence of the glyphosate transporters GltT and GltP does not affect the fitness of the strains!**

To detect glyphosate by intraspecies competition, we mixed the wild type strain synthesizing the GFP protein with the Δ*gltT* Δ*gltP* double mutant synthesizing the mOrange fluorescence protein in a 1:1 ratio. As shown in Figure 20, the amount of wild type cells decreased in the co-culture with increasing glyphosate concentrations. By contrast, the amount of cells of the strain carrying the mOrange gene increased with increasing glyphosate concentrations since the Δ*gltT* Δ*gltP* mutant tolerates the herbicide glyphosate. Thus, **the intraspecies competition assay, which is based on fluorescently labelled *B. subtilis* strains seems to be suitable to detect glyphosate!**

![](https://static.igem.org/mediawiki/2018/e/ec/T--Goettingen--competitionassay_microscopemitauswertungneu_h.png)

**Figure 20.** Green and orange cells represent the wild type strain (*gfp*) and the double transporter mutant (Δ*gltT* Δ*gltP mOrange*), respectively. The ratio indicates the relative amount of orange cells in comparison to green cells. (A) Merged orange and green channels from culture with 0 mM glyphosate after 4 h of cultivation. There are nearly equal amounts of green and orange cells. (B) Merged channels orange and green from culture with 2 mM glyphosate after 4 h of cultivation. The number of orange cells is 4.21-fold higher. (C) Merged orange and green channels from culture with 3.5 mM glyphosate after 4 h of cultivation. Nearly no green cells were visible. (D) Calculated ratio between the number of orange and green cells with increasing glyphosate concentrations.

To perform a competition assay in a greater scale, we used a platereader for our growth experiments. This device enabled us to measure different fluorophores and the optical density simultaneously. Strains were cultivated in CS-glucose (CS-Glu) medium to an OD600 of 1–1.5. A 96–well plate was prepared containing 100 µl of CS-Glc minimal medium with the desired concentrations of glyphosate. 10 µl of the cell mixture (1:1 ratio) were transferred into the wells to obtain an OD600 of 0.1 in the wells. We used the **Synergy MX II** multi-well plate reader from the [BioTek](http://go.biotek.com/readers_de?Product_Interest_Source=PPC%20-%20Adwords&source=PPC%20-%20Adwords&Lead_Campaign_Source=7011O000002tr2g&Campaign_Source=7011O000002tr2g&gclid=Cj0KCQjw6fvdBRCbARIsABGZ-vTKWShl0pOKo23vGKkfvR5HGHyuG-xBZyMQf7Xg6AU56F2M_aH75kIaAnCWEALw_wcB) company for our measurements. GFP was excited at 488 nm and emission was measured at 509 nm. for mOrange, the excitation wavelength was 529 nm and emission was detected at 562 nm.

![](https://static.igem.org/mediawiki/2018/b/bd/T--Goettingen--competitionassay_180911_hi.png)

**Figure 21.** The fluorescence is normed to the optical density, which equals the cell number. **A:** Intensity of GFP fluorescence decreases with inceasing glyphosate concentration(0–2 mM glyphosate). **B:** Intensity of mOrange fluorescence increases with increasing glyphosate concentration 0–2 mM glyphosate).

This competition assay enables us to calculate the concentration of glyphosate in samples from people that are curious, how much glyphosate is in their soil.

#### 4.2. Competition with β–galactosidase expressing strains

Another approach using competition assays is to use enzymatic indication systems. Therefore, we used the strain BP235 (Δ*gltT* Δ*gltP*) and the strain BP193 (*palf4::lacZ*) (12). As previously described, BP235 is characterized by high-level glyphosate resistance, while BP193 resembles the wild type despite the *lacZ* gene. The promoter *palf4* is an artificial one and harbors a perfect consensus sequence for *B. subtilis* and a ribosome binding site. This leads to constitutive expression of the *lacZ* gene and to blue cells if the medium is supplemented with X-Gal. Cultures of both strains in CS-Glc minimal medium without glyphosate were mixed to obtain a 1:1 ratio of both strains and incubated with increasing glyphosate concentrations (0 - 5mM) for 24 h (Figure 22).

![](https://static.igem.org/mediawiki/2018/f/f5/T--Goettingen--competitionassay_blue.png)

**Figure 22.** Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0–5 mM). Strain BP193 harbors the *lacZ* gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235. The intensity of the blue color decreases with increasing glyphosate concentration. Strains BP193 and BP235 were also incubated without glyphosate as a control.

The intensity of the blue color drecreases with increasing glyphosate concentrations (Figure 22). The change and intensity of the blue color correlates with the glyphosate concentration and can therefore be used as detection and quantification system. Furthermore, a β–galactosidase assay can quantify the activity of the promoter *palf4* and thus, the concentration of glyphosate.

The promoter-reporter gene fusion of *palf4* and lacZ allows to determine the activity of the promoter. Since *palf4* is constitutively active this is directly linked to the survival of the cells. Transcription results in synthesis of ß-galactosidase which in turn hydrolyses o-nitrophenyl-ß-D-galactopyranoside (ONPG), resulting in the formation of o-nitrophenol. Formed o-nitrophenol absorbs light with a wavelength of 420 nm, and can therefore be used as an indicator for the amount of cells and subsequently for the concentration of glyphosate. We determined ß-galactosidase activity for the mixture of BP193 and BP235 in presence of increasing amounts of glyphosate. In addition, we used BP193 and BP235 as controls. Cells were grown in a 1:1 ratio. ß-galactosidase activity was high in the BP193 control and at low glyphosate concentration. Increasing glyphosate concentration is directly linked to cell death of BP193 and a corresponding decrease in ß-galactosidase activity (Figure 23).

![](https://static.igem.org/mediawiki/2018/0/07/T--Goettingen--193lacZ.png)

**Figure 23.** Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0 - 5 mM). Strain BP193 harbors the *lacZ* gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235. ß-galactosidase activity can be measured at a wavelength of 420 nm using the substrate o-nitrophenyl-ß-D-galactopyranoside. Activity decreases with increasing glyphosate concentration. BP235 does not contain the *lacZ* gene, hence no activity can be observed.

In an additional step, we verified that the change in coloration occurred due to the death of the sensitive strain on the one hand and the increased growth of the resistant BP235 strain on the other hand. Hence, we adopted the experimental set up and analysed growth of the two strains in the platereader. To analyse growth of BP235, we used the strain constitutively expressing GFP, which was constructed similarly to the previously used strains. For the analysis we again used 100 µL CS-Glu medium supplemented with the appropriate glyphosate concentration. The cells were added in a 1:1 ratio. GFP was excited at 488 nm and emission was measured at 509 nm.

![](https://static.igem.org/mediawiki/2018/d/dc/T--Goettingen--gfp-xgal.png)

**Figure 24.**  Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0–5 mM). Strain BP193 harbors the *lacZ* gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235.

## 5. Engineering bacteria to disarm glyphosate

#### 5.1. The glyphosate *N*-acetyltransferase confers resistance to weedkiller

Many years ago it has been found that glyphosate can be inactivated by acetylation yielding *N*-acetylglyphosate (see Figure 25) (37). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. **The glyphosate *N*-acetyltransferase GAT from the Gram-positive soil bacterium *Bacillus licheniformis* transfers the acetyl group from acetyl-CoA onto the amino group of the weedkiller** (37). Interestingly, *B. subtilis* possesses a putative glyphosate N-acetyltransferase, which is designated as [YitI](http://www.subtiwiki.uni-goettingen.de/v3/gene/view/2751630B6472346B19FB20F57F16013D0FACA9F7) and shares 59% overall amino acid identity with GAT of *B. licheniformis*. However, the native GAT enzymes are very poor catalysts for *N*-acetylation of glyphosate (37).
Moreover, despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (39). As an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to *N*-acetylglyphosate the GAT was also subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide (37). When introduced into plants, optimized *gat* genes confer robust tolerance to glyphosate (37). Therefore, we expect that bacteria like *B. subtilis* expressing the optimized GAT should tolerate high amounts of glyphosate even though it is taken up from the environment *via* the high-affinity glyphosate transporter GltT (see above). To evaluate the potential of the GAT to allow *B. subtilis* in the presence of glyphosate concentrations that are toxic for the wild type bacteria, we wanted to express the *gat* gene in *B. subtilis* (Figure 25).

![](https://static.igem.org/mediawiki/2018/c/ca/T--goettingen--gat_engineering.jpg)

Figure 25. (A) Sequence alignment showing the similarities of the wild type (wt) *gat* alleles from *Bacillus licheniformis* and *Bacillus subtilis* as well as the *gat* alleles R7 and R11 from *B. licheniformis* that were obtained by 11 rounds of gene shuffling. The red arrows indicate the 31 amino acid exchanges in the glyphosate *N*-acetyltransferase (Gat) variant R11. The optimized Gat variants shown an up to a 4,500-fold increase in catalytic efficiency (kcat/Km) relative to the native enzyme. The [GenBank](https://www.ncbi.nlm.nih.gov/genbank/) accession numbers for the sequences: *B. licheniformis* ST401 GAT, [AX543338](https://www.ncbi.nlm.nih.gov/nuccore/AX543338); R7 GAT, [AY597417](https://www.ncbi.nlm.nih.gov/nuccore/AY597417); R11 GAT, [AY597418](https://www.ncbi.nlm.nih.gov/nuccore/AY597418); *B. subtilis* [YitI](http://subtiwiki.uni-goettingen.de/v3/gene/view/2751630B6472346B19FB20F57F16013D0FACA9F7),  [CAA70664](https://www.ncbi.nlm.nih.gov/protein/CAA70664). (B) Structure of the Gat R7 variant (PDBid: [2JDD](https://www.rcsb.org/structure/2jdd)) in ternary complex with acetyl-CoA and the competitive inhibitor 3-phosphoglycerate (3PG). The four active site residues (Arg-21, Arg-73,
Arg-111, and His-138), which are labelled in black color, contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. (C) Top view on the active site of Gat R7. (D) Construction of a plasmid for the expression of the *gat* R11 variant in *B. subtilis*. Expression is driven by the constitutively active σA-dependent *Palf4* promoter The plasmid pAC7::*gat* R11 integrates into the *amyE* locus of the *B. subtilis* chromosome in single copy. *lacZ*, beta-galactosidase; *amyE-5'* and *amyE-3'*, fragments of the *B. subtilis* *amyE* for homologous recombination; *amp*, ampicillin resistance gene for the selection in *E. coli*; *kan*, kanamycin resistance gene for the selection in *B. subtilis*; *ori* pBR322, origin of replication for *E. coli*.

For this purpose, we ordered the *gat* gene as a g-Block from [Integrated DNA Technologies](https://eu.idtdna.com/pages), which was free of charge because the company supports the iGEM competition. Next, we amplified the *gat* gene by PCR using the g-Block as template DNA. An artificial promoter and a ribosome binding site was attached during the PCR (Figure 25D). The promoter-gene fusion was cloned and introduced in single copy into the genomes of the *B. subtilis* wild type strain and the *gltT* mutant strain to further increase its glyphosate tolerance (Figure 7). To assess the potential of the GAT to disarm glyphosate in the different *B. subtilis* strains, we propagated the bacteria on CS-Glc minimal medium agar plates that were supplemented with different amounts of glyphosate. As shown in Figure 26, **the strains synthesizing the GAT tolerated significantly more glyphosate than the wild type strain**. Moreover, the inactivation of the *gltT* glyphosate transporter gene and the overexpression of the *gat* gene confers high-level glyphosate tolerance to the bacteria. Thus, when introduced into *B. subtilis*, the optimized *gat* gene confers indeed robust tolerance to glyphosate. To conclude, **the engineered bacteria possess two interesting properties: (i) *B. subtilis* promotes plant growth by protecting plants against pathogens and (ii), the bacteria take up glyphosate from the environment and inactivate the weedkiller!**

![](https://static.igem.org/mediawiki/2018/d/d3/T--goettingen--roundup_aroA_gat_sp1.jpg)

Figure 26. (A) Growth of the wild type and Δ*gltT* mutant and of the isogenic strains overexpressing the *gat* R11 and *aroA\** genes on CS-Glc minimal medium in the presence of different amounts of glyphosate. (B) Growth of the strains described in A on CS-Glc minimal medium in the presence of different amounts of Roundup® Alphee.

Cultivation of the different strains in liquid media allows more detailed characterization. Therefore, we analyzed growth of the bacteria in CS-Glc minimal medium supplemented with increasing amounts of glyphosate, using a multi-well plate reader. In general, the analysis supports previous results (see Figure 26). The strain containing the *aroA\** gene confers only a slight increase in resistance in comparison to the wild type strain and hence a significantly lower resistance in comparison the *gltT* mutant strain (Figure 27A, B and D). However, for the strain containing the *aroA\** gene and lacking the glyphosate transporter GltT a 9-fold higher glyphosate concentration is needed to reduce the growth rate by 50% (Figure 27F). The growth experiments confirm our previous results showing that the glyphosate resistance in can be strongly enhanced in *B. subtitlis* by expressing the *gat* gene or by inactivating the *gltT*. Moreover, a combination of both even further enhances glyphosate resistance of the bacteria.
(Figure 27C and E, Figure 26).

![](https://static.igem.org/mediawiki/2018/7/76/T--Goettingen--growtharoa-gat.png)

Figure 27. (A) Growth of the *B. subtilis* wild type strain in presence of 0 - 3.5 mM glyphosate. (B) Growth of the *B. subtilis* *gltT* mutant strain with increasing glyphosate concentrations (0-30 mM). (C) Growth of the *B. subtilis* strain producing Gat with increasing glyphosate concentrations (0-30 mM. (D) Growth of the *B. subtilis* strain producing AroA\* with increasing glyphosate concentrations (0-30 mM). (E) Growth of the *B. subtilis* strain producing AroA\* with increasing glyphosate concentrations (0-30 mM). Growth of the  *B. subtilis* strain lacking GltT and producing Gat with increasing glyphosate concentrations (0-30 mM). (F) Growth of the  *B. subtilis* strain lacking GltT and producing AroA\* with increasing glyphosate concentratiosn (0-30 mM). Bacteria were cultivated in liquid CS-Glc minimal medium.

![](https://static.igem.org/mediawiki/2018/6/6c/T--Goettingen--ratearoagat.png )

Figure 28. The relationship between the growth rate (µ) and the glyphosate (GS) concentration for the *B. subtilis* wild type (WT) strain SP1, the Δ*gltT* mutant strain BP233, the *B. subtilis* strain producing Gat, the *B. subtilis* strain producing AroA\*, the *B. subtilis* strain lacking GltT and producing AroA\*, the *B. subtilis* strain lacking GltT and producing Gat in CS-Glc minimal medium supplemented with increasing amounts of GS.

## 6. Characterization of EPSP synthases

#### 6.1. Essentiality of the *B. subtilis* *aroE* EPSP synthase gene

To our surprise, **we did not find glyphosate-resistant mutants of *B. subtilis* that had accumulated mutations in the *aroE* gene rendering the encoded EPSP synthase insensitive to the herbicide**. Since AroE from *B. subtilis* belongs to the class II EPSP synthases, which are less sensitive to glyphosate, the potential of enzyme to further evolve herbicidal insensitivity might be limited (Figure 29) (62-64).

![](https://static.igem.org/mediawiki/2018/0/02/T--goettingen--Stammbaum_icon.jpg)

Figure 29. The EPSP synthase fall into three different groups. While the *E. coli* EPSP synthase glyphosate sensitive (group I), the *B. subtilis* enzyme belongs to the group II of glyphosate-insensitive EPSP synthases. Not much is known about the glyphosate-dependent inhibition of the EPSP synthases that belong to group III. The phylogenetic three has been constructed using the Geneious software package ([Biomatters Ltd.](https://www.geneious.com/about/)). Sequences of the EPSP synthases were derived from the [UniProt](https://www.uniprot.org) database.

Moreover, **experimental evidence suggests that the *B. subtilis aroE* gene is essential for growth even under conditions that do not require the enzymatic function of the EPSP synthase** (65). As essential bacterial genes appear to be more conserved than nonessential genes, the *aroE* gene probably does not permit the accumulation of mutations, which can be beneficial (66). To confirm that the *aroE* gene is indeed essential, we transformed the *B.%nbsp;subtilis* with a PCR product consisting of DNA fragments flanking the target gene and the intervening *spc* spectinomycin gene (*aroE::spc* deletion cassette) and propagated the bacteria on SP rich medium plates (see Notebook). Several tiny colonies appeared on the transformation plates after 24 h of incubation. However, the potential transformants were not viable and supplementation of the agar plates with casamino acids also did not improve growth of the bacteria. Thus, under the tested conditions *aroE* seems to be essential in *B. subtilis*.
Next, we tested whether the chromosomal copy of the *aroE* gene becomes dispensable for the bacteria carrying an EPSP synthase gene on a plasmid. For this purpose, we transformed the *B. subtilis* wild type strain 168 with the plasmids pIGEM2 and pBP143 containing the *aroE* and *aroA* EPSP synthase genes from *B. subtilis* and *E. coli*, respectively. The wild type carrying the empty plasmid pBQ200 served as a control. Next, we transformed the three strains with the *aroE::spc* deletion cassette and with chromosomal DNA of strain BP233 (*gltT::spc*), of which the latter served as the positive control. While we did not get transformants without DNA, many transformants appeared with chromosomal DNA of strain BP233 (Figure 30). Moreover, the chromosomal copy of the *aroE* gene was only dispensable in strains carrying an extra EPSP synthase gene on a plasmid. **To conclude, the *aroE* EPSP synthase gene is essential in *B. subtilis*. Moreover, the bacteria seem to require the enzymatic function of the EPSP synthase because also the enzyme from *E. coli* permits growth of the *aroE* mutant** (Figure 30).

![](https://static.igem.org/mediawiki/2018/1/19/T--goettingen--aroE_essentiality.jpg)

Figure 30. The EPSP synthase is essential in *B. subtilis*.
Transformation experiment to evaluate the essentiality of the EPSP synthase in *B. subtilis*. The bacteria were propagated on SP plates supplemented with spectinomycin and incubated for 24 h at 37°C.

#### 6.2. Purification and characterization of EPSP synthases

This year, our team came toghether with the [iGEM team of Marburg](https://2018.igem.org/Team%3AMarburg), to investigate the interaction of the EPSP synthases AroE and AroA from *B. subtilis* and *E. coli*, respectively, with glyphosate. Glyphosate targets AroE and AroA in *B. subtilis* and *E. coli*, respectively (see Background information). Since the members of the [Bange lab](https://www.bangelab.org), which is hosting the [iGEM team of Marburg](https://2018.igem.org/Team%3AMarburg), are experts in the field of
[Isothermal titration calorimetry](https://en.wikipedia.org/wiki/Isothermal_titration_calorimetry) (ITC), we did not hesitate to contact them for support. Our team cloned the *aroE* and *aroA* genes of *B. subtilis* and *E. coli*, respectively, and **the resulting constructs are suitable for overexpression of the
Strep-tagged AroE and AroA enzymes in the *E. coli* strain BL21**. The N-terminally Strep-tagged proteins can be purified by Streptactin:Strep-tag affinity purification system from the
[IBA](https://www.iba-lifesciences.com/home.html?gclid=CjwKCAjwlejcBRAdEiwAAbj6Kbo80UFPjKO6Yzdy03cr_0LVHUcV7ZbYCE-x-HBVWXZY9-_pbBP4QhoCrfAQAvD_BwE), Göttingen. The purified proteins were send to the iGEM team in Marburg (Figure 31). The ITC measurments were performed in Marburg. Unfortunately, not interaction between glyphosate and the EPSP synthases could be detected. Unfortunately, also the second ITC measurement using freshly purified AroA and AroE enzymes from *E. coli* and *B. subtilis*, respectively, did not reveal whether glyphosate may interact with the EPSP synthases. Nevertheless, we are grateful to the iGEM Team Marburg for performing the ITC measurements.

![](https://static.igem.org/mediawiki/2018/1/18/T--goettingen--aroE_aroA_Strep_ITC_Marburg.jpg)

Figure 31. (A) Evaluation of the purification of the N-terminally Strep-tagged EPSP synthases from *B. subtilis* and *E. coli* Strep-AroE and Strep-AroA, respectively, by 12% SDS PAGE. The proteins were stained with Coomassie Brilliant Blue. M, unstained protein molecular weight marker [Thermo Scientific](https://www.thermofisher.com/de/de/home/brands/thermo-scientific/molecular-biology.html); CE, crude extract; FT, flow through, W, washing steps; E, elution steps. (B) ITC measurements with Strep-AroE and Strep-AroA and glyphosate. (C) Control experiments with water and the calcium chelator EDTA.

---

## References

1. Fischer *et al.* (1986) J. Bacteriol. 168: 1147-1154- Zeigler *et al.* (2008) J. Bacteriol. 190: 6983-6995.- Barbe *et al.* (2009) Microbiology. 155: 1758-1775- Kearse *et al.* (2012) Bioinformatics. 28: 1647-1649.- Omasits *et al.* (2014) Bioinformatics. 30: 884-886.- Michna *et al.* (2014) Nucleic Acids Res. 42: D692–D698.- Zaprasis *et al.* (2015) Appl. Environ. Microbiol. 81: 250-259.- Tolner *et al.* (1995) J. Bacteriol. 177: 2863-2869.- Amrhein *et al.* (1980) Naturwissenschaften. 67: 356-357.- Comai *et al.* (1983) Science. 221: 370-371.- Stalker *et al.* (1985) J. Biol. Chem. 260: 4724-4728.- Gundlach *et al.* (2017) Sci. Signal. 10: eaal3011.- Gundlach *et al.* (2017) Sci. Signal. 10: eaal3011.- Fraser & Ridley (1984) Planta. 161: 470-474.- Priestmann et al., (2005) FEBS Lett. 579: 728-732.- Cao *et al.* (2012) PLoS One. 7: e38718.- Light *et al.* (2016) Biochemistry. 55: 1239-1245.- Liu & Cao, (2018) Biotechnol. Lett. 40: 855-864.- Rogers *et al.* (1983) Appl. Envrion. Microbiol. 46: 37-43.- Gaines *et al.* (2011) J. Agric. Food Chem. 59: 5886-5889.- Juglam *et al.* (2014) Plant Physiol. 166: 1200-1207.- Sammons & Gaines (2014) Pest. Manag. Sci. 70: 1367-1377.- Dillon *et al.* (2017) Plant Physiol. 173: 1226-1234.- Sost & Amrhein (1990) Arch. Biochem. Biophys. 282: 433-436.- Padgette *et al.* (1991) J. Biol. Chem. 266: 22364-22369.- He *et al.* (2001) Biochim. Biophys. Acta 1568: 1-6.- He *et al.* (2003) Biosci. Biotechnol. Biochem. 67: 1405-1409.- Bearson *et al.* (2002) Plant Physiol. 129: 1265-1275.- Eschenburg *et al.* (2002) Planta 216: 129-135.- Sun *et al.* (2005) Appl. Environ. Microbiol. 71: 4771-4776.- Zhou *et al.* (2006) Plant Physiol. 140: 184-195.- Healy-Fried *et al.* (2007) J. Biol. Chem. 282: 32949-32955.- Vande Berg *et al.* (2008) Pest. Manag. Sci. 64: 340-345.- Funke *et al.* (2009) J. Biol. Chem. 284: 9854-9860.- Tian *et al.* (2010) Appl. Environ. Microbiol. 76: 6001-6005.- Pollegioni *et al.* (2011) FEBS J. 278: 2753-2766.- Chekan *et al.* (2016) MedChemComm. 7: 28-36.- Castle *et al.* (2004) Science. 304: 1151-1154.- Siehl *et al.* (2005) Pest. Manag. Sci. 61: 235-240.- Siehl *et al.* (2007) J. Biol. Chem. 282: 11446-11455.- Norris *et al.* (2009) Appl. Environ. Microbiol. 75: 6062-6075.- Rao *et al.* (1983) Antimicrob. Agents Chemother. 24: 689-695.- Penaloza-Vazquez *et al.* (1995) Appl. Environ. Microbiol. 61: 538-543.- Liu *et al.* (2015) Transgenic Res. 24: 753-763.- Staub *et al.* (2012) J. Ind. Microbiol. Biotechnol. 39: 641-647.- Tao *et al.* (2017) Pestic. Biochem. Physiol. 140: 65-68.- Fitzgibbon & Braymer, (1988) Appl. Environ. Microbiol. 54: 1886-1888.- Fitzgibbon & Braymer, (1990) Appl. Environ. Microbiol. 56: 3382-3388.- Fartyal *et al.* (2018) Front. Plant. Sci. 13: 144.- Pipke *et al.* (1980) Appl. Environ. Microbiol. 53: 974-978.- Shinabarger *et al.* (1986) J. Bacteriol. 168: 702-707.- Liu *et al.* (1991) Appl. Environ. Microbiol. 57: 1799-1804.- Dick & Quinn, (1995) Appl. Microbiol. Biotechnol. 43: 545-550.- Singh & Walker, (2006) FEMS Microbiol. Rev. 30: 428-471.- Castro *et al.* (2007) J. Environ. Sci. Health B. 42: 883-886.- Hove-Jensen *et al.* (2014) Microbiol. Mol. Biol. Rev. 78: 176-197.- Kryuchkova *et al.* (2014) Microbiol. Res. 169: 99-105.- Sviridov *et al.*, (2011) Biochemistry (Mosc). 76: 720-725.- Sviridov *et al.* (2015) Prikl. Biokhim. Mikrobiol. 51: 183-190.- Lu *et al.* (2013) Mol Biosyst. 9: 522–30.- Gaballa & Helmann (1998) J. Bacteriol. 180: 5815–5821.- Gaballa *et al.* (2002) J. Bacteriol. 184: 6508–6514.- Barry *et al.* (1992) WO 92/04449.- Schönbrunn *et al.* (2001) Proc. Natl. Acad. Sci. USA. 98: 1376-1380.- Light *et al.* (2016) Biochemistry. 66: 1239-1245.- Koo *et al.* (2017) Cell Systems. 4: 291-305.- Jordan *et al.* (2002) Genome Res. 12: 962-968.