iGEM 2018 — Team HSHL

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# Team:HSHL/Experiments

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[Design](https://2018.igem.org/Team%3AHSHL/Design)
[Experiments](https://2018.igem.org/Team%3AHSHL/Experiments)
[Results](https://2018.igem.org/Team%3AHSHL/Results)
[Attributions](https://2018.igem.org/Team%3AHSHL/Attributions)

[PARTS](https://2018.igem.org/Team%3AHSHL/Parts)
[SAFETY](https://2018.igem.org/Team%3AHSHL/Safety)
[HUMAN PRACTICES](https://2018.igem.org/Team%3AHSHL/Human_Practices)

AWARDS

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

[CONTACT & COPYRIGHT](https://2018.igem.org/Team%3AHSHL/ContactCopyright)
[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=HSHL)

# Protocols & Experiments

In the following you will find a description of our experiments, and protocols used in our iGEM project. You want to repeat our experiments and need further information? [Contact us](https://2018.igem.org/Team%3AHSHL/ContactCopyright), we are glad to help you.

### Quicklinks

* [RIM-Medium](#rim)
* [SOC-Medium](#soc)
* [Liquid LB-Medium](#lb_liq)
* [Liquid LB-Medium](#lb_sol)
* [Gel electrophorasis](#gel_elektro)
* [Agarose](#agarose)
* [Transformation via heat shock](#heat)
* [Cloning Strategy](#cloning)

### Inactivationtemperatures

|  | Temp. [°C] | Time [min] |
| --- | --- | --- |
| T4 DNA Ligase (NEB) | 65 | 10 |
| EcoRI (NEB) | 65 | 15 |
| PstI (NEB) | 80 | 20 |
| Shrimp Alkaline Phospatase (NEB) | 65 | 5 |
| HindIII (NEB) | 80 | 20 |
| EcoRII (Thermofisher Scientific) | 80 | 20 |
| SmaI (NEB) | 65 | 20 |

We used 3 heat baths: 1 with 80°, 1 with 65°, 1 with 37°

For heat shock: increased temperature of the 37° bath to 42°

**- RIM-Medium for plant cultivating**

1. „Murashige” + Skoog Medium including Vitamins and MES Buffer (concentration: 4.9 g/L)
2. Adding Saccharose
    2 % (w/v)  2 g/100 mL = 20 g/L
3. Adding Agar Type M
    0.7 – 0.8 % (w/v)  0.7 – 0.8 g/100 mL = 7 – 8
   g/L
4. Adjust pH of the medium with help of KOH (Kaliumhydroxide)
    pH 5.7 – 5.8
5. Autoclaving of this liquid for 10 minutes for sterilizing
6. Cool down of autoclaved medium (ca. 50 – 55 °C)
7. Preparing IES stock solution
    1mg IES / 1 mL Ethanol  scale and then
   storing at 4 °C
8. For 500 mL medium 500 µl IES Stocklösung sterile in medium pipette
     for
   1 L medium put 1 mL stock
   solution in medium

**- SOC-Medium**

1. We used NEB SOC-Medium and prepared it according to manufacturer’s instructions

**- LB-Medium (1L liquid)**

1. 10 g tryptone
2. 10 g NaCl
3. 5 g yeat extract
4. Water
5. (in case we needed chloramphenicol-medium, we used 50 µl for 50 ml medium)

**- LB-Medium (solid, 1L = 50 dishes)**

1. 15 g agar agar
2. 10 g tryptone
3. 10 g NaCl
4. 5 g yeat extract
5. Water
6. (in case we needed chloramphenicol-medium, we used 50 µl for 50 ml medium)

**- Gel-electrophorasis protocol**

1. Pour 1x TAE buffer in electrophoresis chamber, until the agarosegel is covered by approximately 5mm.
2. Remove agarosegel pocket spacer
3. Add 12 µl of marker into a pocket
4. Close electrophoresis chamber and connect to power supply.
5. Set power supply to 100V, after 5 minutes set voltage to 200V. Run electrophoresis until the marker is
   approximately 1cm from the bottom of the gel (approximately 30 min)
6. Swich off power supply, remove gel and stain it with staining solution (Sybr Safe). Afterwards wash gel and
   analyse bars.

**- Agarose**

* Agarose
* TAE 50x - Buffer
* ddH20

1. Prepare 30 mL of 2% Agarose solution. To do so calculate Agarose concentration by (w/v) and weight the
   Agarose.
2. Add 7 mL of 50x TAE buffer into a new flask and fill with ddH2O to 350mL.
3. Seal flask with Parafilm and mix. TAE buffer is now 1x TAE buffer.
4. Place gel pad into electrophoresis chamber and place and add gel barriers into chamber. Afterwards place
   pocket spacer into gel pad.
5. Add 30mL of 1x TAE buffer to Agarose and mix.
6. Heat Agarose & 1x TAE buffer solution in microwave until solution is clear
7. Pour Solution into electrophoresis chamber consistently
8. Let gel cool for 20 min

**- Transformation via heat shock**

1. Thaw 200 μL chemocompetent E. Coli cells on ice
2. Add 0.5-5 μL plasmid to 200 μL chemocompetent ells
3. Store cells on ice for 10-30 min
4. Heatshock for 90 s at 42°C
5. Transfer transformation reaction to 1mL SOC-Medium and incubate 1 h at 37°C
6. Centrifuge 30 s at 11000 rpm and plate on selective LB-Medium
7. Incubate overnight at 37°C

**- Cloning Strategy with restriction enzymes**

* X μl 600ng Vektor
* 3 μl 10x buffer (NEB2.1)
* 1 μl 1. restriction enzyme
* 1 μl 2. restriction enzyme
* Add 30 μl dd H2O

1. Digestion: Vector/Backbone

    Incubation 1 h, 37°C

    Inactivation 10 min, 37°C

* X μl 100ng Insert
* 3 μl 10x buffer
* 1 μl 1. restriction enzyme
* 1 μl 2. restriction enzyme
* Add 30 μl dd H2O

2. Digestion: Insert

    Incubation 1 h, 37°C

    Inactivation 10 min, 37°C

3. Dephosphorylation of Vector/Backbone

    add 2 μL of Shrimp Alkaline Phosphatase (Jena Bioscience)

    Incubation 30 min, 37 °C

    Inactivation 5 min, 75 °C
4. Phosphorylation of Insert

    add 3 μL 10x T4 Ligase Buffer (Thermo Fisher) -> contains right amount of ATP

    add 1 μL PNK (NEB)

    Incubation 30 min, 37 °C

    Inactivation 20 min, 65 °C
5. Purification of Vector and Insert

    Any gicen Gel an PCR Purification Kit

    We recommend purification after gel electrophoresis

This wiki is designed by HSHL within the iGEM 2018 template.

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# Team:HSHL/Results

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ABOUT US

[Team](https://2018.igem.org/Team%3AHSHL/Team)
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[Collaborations & Networking](https://2018.igem.org/Team%3AHSHL/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AHSHL/Description)
[Design](https://2018.igem.org/Team%3AHSHL/Design)
[Experiments](https://2018.igem.org/Team%3AHSHL/Experiments)
[Results](https://2018.igem.org/Team%3AHSHL/Results)
[Attributions](https://2018.igem.org/Team%3AHSHL/Attributions)

[PARTS](https://2018.igem.org/Team%3AHSHL/Parts)
[SAFETY](https://2018.igem.org/Team%3AHSHL/Safety)
[HUMAN PRACTICES](https://2018.igem.org/Team%3AHSHL/Human_Practices)

AWARDS

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

[CONTACT & COPYRIGHT](https://2018.igem.org/Team%3AHSHL/ContactCopyright)
[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=HSHL)

# Results

The synthesis of the HMA3 gene which was needed for this experiment was done by IDT. A gene with the entire sequence was synthesized as well as a gene that was divided into four fragments.
First, Gen and Vector were prepared for ligation by cutting both components using the enzymes EcoRI and PstI. The vector, called pSB1C3 was then dephosphorilised by Shrimp Alkaline Phosphatase and the insert, the gene HMA3, phosphorilised by T4 Polynucleotide Kinase. After that gel electrophoresis and the Wizard SV Gel and PCR Clean- Up System of the company Promega were used to purify both components. Below you can see the result of the gel electrophoresis purification of the four fragments.

The IDT 2- log DNA ladder was used as a control. The following figure shows the Ladder schematically.

By evaluating the gel electrophoresis results with the help of the ladder, the following quantities results are determined for the four fragments.

| Fragment No. | Length [bp] |
| --- | --- |
| 1 | 614 |
| 2 | 593 |
| 3 | 556 |
| 4 | 885 |

After purification using gel electrophoresis, the bands were cut out and the DNA was extracted from the gel using the promega Wizard SV Gel and PCR Clean- Up System.

Then vector and insert could be ligated in the following ratio.

| Component | Amount [µL] |
| --- | --- |
| Insert | 8.15 |
| Vector | 5.33 |
| T4 Ligase Buffer | 2.00 |
| T4 Ligase | 1.00 |
| H20 | 13.52 |

After the insert and the vector were ligated the transformation could be started. For this experiment, 200µL chemocompetent *E. coli DH5 α* were mixed with 5µL of the ligated plasmid. We used a heat shock to get the plasmid into the cells. The heat shock was performed at 42 degrees for 90 seconds after the cells were stored on ice for 30 minutes. The cells were then first stored in SOC medium for one hour and then applied to LB- Agar with chloramphenicol for incubation.

In order to make sure that the individual steps of the experiment worked, several further experiments with positive controls were carried out. We have made a positive control of the used *E. coli DH5 α* by applying them to an LB- Agar without chloramphenicol. The following figure shows the result of the positive control.

*Figure 3* shows that *E. coli* cells grew on the LB agar without chloramphenicol. This means the cells we had used for our project were in a vital state. For this reason, there is no concern to use the cells for further experiments.

![](https://static.igem.org/mediawiki/2018/b/b9/T--HSHL--gelImg.png)
**Fig. 1**: Result of gel electrophoresis purification of the four fragments

![](https://static.igem.org/mediawiki/2018/7/73/T--HSHL--idt2-logLadder.jpg)
**Fig. 2**: Representation of the IDT 2-log ladder and the length of its bands

![](https://static.igem.org/mediawiki/2018/d/dd/T--HSHL--EcoliPosControl.jpg)
**Fig. 3**: Result of positive control of the used *E. coli DH5 α*

To make sure that the transformation worked and that the plasmid was absorbed into the cells, a positive control was also performed for this part of the experiment. For this purpose the plasmid provided by the IGEM headquarter was transformed into the cells. The used plasmid is called pSB1C3. The result of this transformation is shown in *Figure 4*.

The figure shows that colonies of transformed *E. coli* cells have grown on the agar with chloramphenicol. That means the plasmid was absorbed into the cells. The pink colonies contain an insert that was used in the production of the plasmid at the iGEM headquarter. Minipreps were then produced from the individual colonies to isolate the plasmid from the cells. For this the Promega Wizard® Plus SV Minipreps DNA Purification System was used. *Figure 5* shows how the minipreps are carried out.

After the minipreps were finished, a gel electrophoresis of the isolated plasmids was performed again. The IDT 2- log DNA ladder was also used for this gel electrophoresis. The gel picture is shown in *Figure 6*.

Now that these further experiments have been successfully completed, the work can continue on the construction of the biobrick. As shown in the previous results, the *E. coli DH5 α* and the vector pSB1C3 are well suited for the following experiments.

![](https://static.igem.org/mediawiki/2018/0/03/T--HSHL--transformationPosControl.jpeg)
**Fig. 4**: Result of positive control of the transformation

![](https://static.igem.org/mediawiki/2018/e/e2/T--HSHL--washingStep.jpg)
**Fig. 5**: Washing step during the miniprep production

![](https://static.igem.org/mediawiki/2018/9/97/T--HSHL--gel02.png)
**Fig. 6**: Result of gel electrophoresis of the positive control colonies

This wiki is designed by HSHL within the iGEM 2018 template.