iGEM 2021 — Team Stuttgart

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# Team:Stuttgart/Notebook

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Notebook
Notebook

# Wet lab notebook

---

---

![](https://static.igem.org/mediawiki/2021/3/39/T--Stuttgart--lab1.png)
## June

---

**29.06.**

Present: Stefanie, Lina

Lab Work:

1 T75 bottle with HeLa received from the Institute of Cell Biology and Immunology.

---

**30.06.**

Present: Stefanie, Lina

Lab Work:

Transferred to our incubator (37°C and 5% CO2).

Medium accepted.

3 ml trypsin and EDTA, incubated for 7 minutes in the incubator.

Supernatant distributed on tubes and centrifuged.

Pellet dissolved in medium.

In T175 bottle with 17 ml medium.

---

---

## July

---

**01.07.**

Present: Sumohit, Ida, Miguel, Ben

Lab Work:

Production of agar medium and LB medium.

---

**02.07.**

Present: Stefanie, Lina

Lab Work:

HeLa cells split from 1 T175 to 3 T175.

like 30.06.

---

**07.07.**

Present: Stefanie, Lina

Lab Work:

HeLa cells split from 1 T175 to 3 T175.

like 30.06.

---

**09.07.**

Present: Stefanie, Lina

Lab Work:

Contamination in HeLa incubator.

---

**13.07.**

Present: Stefanie, Luisa

Lab work:

HeLa cells splitting.

---

**14.07.**

Present: Sumohit, Ida, Miguel, Ben

Lab Work:

Processing of the ordered primers and fragments for the *E. coli* fragments and primers.

PCR with wildtype dsup fragment (F1), C-terminal dsup fragment (F2), N-terminal dsup fragment (F3) and pBAD18.

---

**15.07.**

Present: Sumohit, Ida, Ben, Stefanie, Lina

Lab Work:

Preparation of agarose.

Gel electrophoresis at 100 V, 75mA and 45 minutes.

Cleaning of incubator.

New HeLa cells fetched.

Preparation of trypsin.

---

**16.07.**

Present: Sumohit, Ida,Lina, Luisa

Lab Work:

DNA gel extraction from the agarose gel from 15.07.

Gibson assembly.

DNA clean-up and concentration.

HeLa cells splitting.

---

**19.07.**

Present:Sumohit, Ben, Miguel

Lab Work:

Transformation into DH5α competent cells.

Preparation of Ampicillin solution.

---

**20.07.**

Present: Stefanie, Lina

Lab work:

HeLa cells splitting.

---

**21.07.**

Present:Sumohit, Ida, Miguel

Lab Work:

No colonies on the agar plates.

PCR with F1, F2, F3 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

---

**22.07.**

Present: Sumohit, Ida

Lab Work:

DNA gel extraction from the agarose gel from 21.07.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

pBAD18 plasmid isolation.

---

**23.07.**

Present: Sumohit, Miguel, Ben, Lina

Lab work:

No colonies on the agar plates.

HeLa cells splitting.

---

**27.07.**

Present: Sumohit, Miguel, Stefanie, Ben

Lab Work:

HeLa cells splitting.

For HeLa Gibson:

Processing of the ordered primers and fragments for the HeLa fragments and primers.

For *E. coli* Gibson:

No colonies on the agar plates.

PCR with F1, F2, F3, pBAD18, fragment for HeLa (FHL) and pcDNA3.1.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

---

**28.07.**

Present: Sumohit, Miguel, Ben

Lab Work:

For HeLa Gibson:

PCR with fragment for HeLa (FHL) and pcDNA3.1.

For *E. coli* Gibson:

DNA gel extraction from the agarose gel from 27.07.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

**29.07.**

Present: Sumohit, Miguel

Lab Work:

For *E. coli* Gibson:

No colonies on the agar plates.

---

**30.07.**

Present: Stefanie, Lilli, Sumohit, Miguel

Lab Work:

HeLa cells splitting.

For HeLa Gibson:

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

---

## August

---

**03.08.**

Present:Ben, Lilli

Lab work:

HeLa cells splitting.

---

**04.08. and 05.08.**

Present: Sumohit, Ben

Lab Work:

For *E. coli* Gibson:

PCR with F1, F2, F3 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

**05.08.**

Present: Stefanie, Lilli

Lab work:

Several bottles split, bem rest medium changed.

Seeding: 2 ml each with 20,000-50,000 cells.

Counting chamber: count corner chambers --> mean value

---

**06.08.**

Present: Sumohit, Lilli

Lab Work:

HeLa cells splitting.

For HeLa Gibson:

Plasmid isolation.

Nanodrop --> Concentration is too low for sequencing.

DNA clean-up and concentration for better concentration.

ent for sequencing.

Sample names:

P1F --> Plasmid pcDNA3.1 1st with forward Primer

P1R --> Plasmid pcDNA3.1 1st with reverse Primer

P2F --> Plasmid pcDNA3.1 2nd with forward Primer

P2R --> Plasmid pcDNA3.1 2nd with reverse Primer

14:00 to Mr Eisler --> 1 sample was set under microscope- setting of 2 blocks: 1st block normal light 48 hours; 2nd Blocj UV 7 sec all for 24 hours minimal.

---

**09.08.**

Present: Sumohit, Ben

Lab work:

Plasmid irradiation test.

Colony PCR.

---

**10.08.**

Present: Sumohit, Ben, Stefanie

Lab work:

HeLa cells splitting.

Plasmid isolation.

Plasmid irradiation test.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

Colony PCR.

---

**11.08.**

Present: Sumohit, Miguel

Lab work:

Production of agar medium and LB medium.

PCR for plasmid isolation test.

---

**12.08 and 13.08.**

Present: Sumohit, Miguel, Lilli

Lab Work:

HeLa cells splitting.

For *E. coli* and HeLa Gibson (From now with NEBuilder):

PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

**16.08 and 17.08.**

Present: Sumohit, Ben, Stefanie, Lina

Lab Work:

HeLa cells splitting.

Colony-PCR with colonies on the agar plates from 12.08 and 13.08

Begin of new live-cell imaging.

For *E. coli* and HeLa Gibson:

PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

**18.08. and 19.08.**

Present: Stefanie, Lina

Lab Work:

HeLa cells splitting.

For *E. coli* and HeLa Gibson:

PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

Begin of new live-cell imaging.

---

**20.08.**

Present: Sumohit, Ida

Lab work:

Production of competent cells.

Transformation into DH5α competent cells.

Plasmid irradiation experiment.

---

**23.08.**

Present: Stefanie, Lina, Lilli

Lab work:

Colony-PCR with colonies on agar plate form 18. and 19.08.

Production of competent cells.

Preparation of Ampicillin solution.

---

**24.08.**

Present: Stefanie, Lina, Lilli

Lab work:

HeLa cells splitting.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

---

**27.08.**

Present: Stefanie, Lina, Lilli

Lab work:

HeLa cells splitting.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

---

**31.08.**

Present: Sumohit, Miguel, Lina

Lab work:

HeLa cells splitting.

Plasmid irradiation experiment.

PCR with irradiated fragments.

---

---

## September

---

**02.09.**

Present: Stefanie, Lina

Lab work:

HeLa life cell imaging.

---

**06.09.**

Present: Sumohit, Lina

Lab work:

Production of agar medium and LB medium.

---

**07.09.**

Present: Sumohit, Ben

Lab work:

HeLa cells splitting.

Plasmid isolation.

For *E. coli* Gibson:

PCR with F1, F2, F3 and pBAD18.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

---

**08.09.**

Present: Sumohit, Ida, Ben

Lab work:

Plasmid irradiation experiment.

PCR with irradiated fragments.

Production of agar medium.

Plasmid isolation from overnight culture with single colonies from the agar plates of 07.09.

Measurement of DNA concentration with Nanodrop.

Sent for sequencing.

Sample names:

Dsup WT

Dsup C-Term

Dsup N-Term

---

**10.09.**

Present: Ben, Stefanie, Sumohit

Lab work:

HeLa cells splitting.

For HeLa Gibson:

PCR with FHL and pcDNA3.1.

Preparation of agarose.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

For *E. coli*:

Transformation into BW25113 competent cells.

Production of TB salts and TB medium.

---

**13.09.**

Present: Ben, Stefanie, Sumohit

Lab work:

HeLa cells splitting.

DNA gel extraction from the agarose gel.

Gibson assembly.

DNA clean-up and concentration.

Transformation into DH5α competent cells.

For *E. coli*:

Preparation for the expression of wildtype dsup and N-terminal dsup.

---

**14.09.-15.09.**

Present: Sumohit, Ben

Lab work:

Expression of wildtype dsup and N-terminal dsup.

---

**16.09.**

Present: Sumohit, Lina

Lab work:

Harvest cells and store in saline until cell breaking.

SDS-PAGE.

---

**14.09.**

Present: Lilli, Lina

Lab work:

HeLa cells splitting.

---

**17.09.**

Present: Lina, Sumohit

Lab work:

HeLa cells splitting.

Preparation for the expression of genotypes for *E. coli*.

SDS-PAGE.

Plasmid isolation for HeLa Gibson.

---

**20.09.-24.09.**

Present: Sumohit, Lina

Lab work:

Expression of genotypes for *E. coli*.

Harvest cells and store in saline until cell breaking.

On 24.09. SDS-PAGE.

---

**21.09.**

Present: Ben, Stefanie

Lab work:

HeLa cells splitting.

---

**24.09.**

Present: Lilli, Ben

Lab work:

HeLa cells splitting.

---

**26.09.**

Present: Sumohit, Lina

Lab work:

Harvest cells and store in saline until cell breaking.

SDS-PAGE.

---

**28.09.**

Present: Lina, Lilli, Sumohit

Lab work:

HeLa cells splitting.

Preliminary tests for intracellular plasmid irradiation.

PCR with FHL and pcDNA3.1.

Preparation of agarose gel.

Gel electrophoresis at 100 V, 75 mA and 45 minutes.

---

**30.09.**

Present: Sumohit

Lab work:

Cell disruption with high pressure homogeniser.

---

---

## October

---

**01.10.**

Present: Lilli, Stefanie

Lab work:

HeLa cells splitting.

---

**04.10.**

Present: Lilli, Sumohit

Lab work:

HeLa cells splitting.

Protein purification in Äkta.

Production of agar medium.

Preliminary tests for intracellular-located dsup cells irradiation.

![](https://static.igem.org/mediawiki/2021/a/a3/T--Stuttgart--%C3%A4kta.png)

---

**05.10.**

Present: Lilli, Sumohit

Lab work:

Expression of N-terminal dsup by 30°C.

Preliminary tests for intracellular-located dsup cells irradiation.

---

**06.10.**

Present: Lilli, Sumohit

Lab work:

Harvest cells and cell disruption with high pressure homogeniser.

---

**08.10.**

Present: Lilli, Sumohit

Lab work:

HeLa cells splitting.

Protein purification in Äkta.

Experiment for intracellular-located dsup cells irradiation.

SDS-PAGE.

![](https://static.igem.org/mediawiki/2021/c/c3/T--Stuttgart--lab2.png)

---

**11.10.**

Present: Lilli, Ben

Lab work:

OD measurement and SDS-PAGE of all expression samples.

---

**12.10.**

Present: Sumohit, Ida

Lab work:

HeLa cells splitting.

SDS-PAGE.

Experiment for intracellular-located dsup cells irradiation.

---

**13.10.**

Present: Lilli, Luisa, Marcel

Lab work:

Bradford assay.

Experiment for intracellular-located dsup cells irradiation.

Preparation of LB medium.

---

**14.10.**

Present: Lilli, Ida, Ben

Lab work:

SDS-PAGE

Preparation for experiment for extracellular-located dsup irradiation.

---

**15.10.**

Present: Lilli, Luisa, Ida

Lab work:

SDS-PAGE.

Experiment with extracellular-located dsup irradiation.

Experiment with intracellular-located dsup irradiation.

---

**18.10.**

Present: Lilli, Stefanie

Lab work:

Sterile filtered sample C-term peak 2

Bradford assay.

HeLa cells splitting.

Experiment for extracellular-located dsup irradiation.

Preparing overnight culture.

---

**19.10.**

Present: Lilli, Lina

Lab work:

Experiment for intracellular-located dsup irradiation

SDS-PAGE.

---

**20.10.**

Present: Lina, Lilli

Lab work:

Experiment for extracellular-located dsup irradiation.

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# Team:Stuttgart/Engineering

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Engineering Success
Engineering Success

## Idea

The Plasmid should contain the Dsup sequence and a His-tag. The addition of a tag could disturb the interaction of Dsup with the DNA. As a result the His-tag was inserted at the C-Terminus and the N-Terminus. We worked with a optimized sequence for E.coli to reduce the risks of unwanted influences.
For the HeLa plasmid, we decided to construct only one fragment with a His-tag on the N-Terminus.

A vector with dsup and His-Tag and without the addition of the His-tag was designed
The dsup sequence can be found on database (https://www.ncbi.nlm.nih.gov/protein/BAV59442.1 retrieved on 15.06.21) and codon optimized for the work with Escherichia Coli and HeLa-Cells by using the "Codon Optimization Tool" from IDT. According to the rules of iGEM, the fragments should not contain any restriction sites that are part of the BioBricks. This has already been taken into account in the cause of the optimization.
Snapgene was used to insert the gene into a suitable plasmid and determine primers for the Gibson assembly. Four dsup constructs were designed, three for E. coli (N-His; C-His and wt) and one for HeLa. The backbones pBAD18 (E. coli) and pcDNA3.1 (HeLa) were used. The part BBa\_K3780011 (wild-type Dsup) contains the dsup, the prefix BioBrick and the suffix BioBrick as well as the arabinose promoter and the promoter region. BBa\_K3780017 (N-terminal His-tag Dsup) contains the same parts but the His-tag is located behind the ATG-Codon. In BBa\_K3780013 (C-terminal His-tag Dsup) the His-tag can be found in front of the stop codon. The dsup that was optimized for HeLa has a His-tag in the N-terminus (BBa\_K3780019).
All sequences for the fragments and primers can be found in the Wet-Lab section.

![Fig. 1: Vector map of empty vector pBAD18 shown by Snapgene.](https://static.igem.org/mediawiki/2021/7/75/T--Stuttgart--pBAD18_LV.jpeg)

Fig. 1: Vector map of empty vector pBAD18 shown by Snapgene.

![Fig. 2: Vector map of wildtype dsup with both BioBricks optimized for E. coli, shown by Snapgene. ](https://static.igem.org/mediawiki/2021/d/db/T--Stuttgart--dsup_WT.jpeg)

Fig. 2: Vector map of wildtype dsup with both BioBricks optimized for E. coli, shown by Snapgene.

![Fig. 3: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for E. coli, shown by Snapgene. ](https://static.igem.org/mediawiki/2021/b/bc/T--Stuttgart--dsup_NTerm.jpeg)

Fig. 3: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for E. coli, shown by Snapgene.

![Fig. 4: Vector map of C-terminal dsup with both BioBricks and His tag in C-terminus optimized for E. coli, shown by Snapgene. ](https://static.igem.org/mediawiki/2021/7/78/T--Stuttgart--dsup_CTerm.jpeg)

Fig. 4: Vector map of C-terminal dsup with both BioBricks and His tag in C-terminus optimized for E. coli, shown by Snapgene.

![Fig. 5: Vector map of empty vector pcDNA3.1(+) shown by Snapgene.](https://static.igem.org/mediawiki/2021/6/63/T--Stuttgart--pcDNA3.1%28%2B%29.jpeg)

Fig. 5: Vector map of empty vector pcDNA3.1(+) shown by Snapgene.

![Fig. 6: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for HeLa, shown by Snapgene.](https://static.igem.org/mediawiki/2021/e/ec/T--Stuttgart--dsup_HeLa.jpeg)

Fig. 6: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for HeLa, shown by Snapgene.

## Cloning and Expression

After the fragments arrived, we started the cloning process. Our E. coli fragments contain an arabinose promoter. The arabinose promoter is a tight promoter, which means that in the absence of arabinose, dsup cannot be expressed, so we can control the expression. We used the Gibson assembly cloning method because it is easier and cheaper than the other methods. The first transformation took places for all fragments in DH5α, because this E. coli strain is very suitable for transformation due to its high transformation efficiency. The second transformation was performed only for the E. coli fragments in BW25113. E. coli strain BW25113 is arabinose-deficient, which is advantageous for our case, as we induce with arabinose and can thus be sure that the E. coli cells do not consume arabinose. Since we did not know at which temperature dsup folds best, we performed the expression at 3 different temperatures: 37°C, 30°C and room temperature. Unfortunately, we did not have enough time to see at which temperature the protein folds best, so we decided to continue with the protein that had been expressed at 30°C.

## Experiments

In order to confirm that Dsup protects cells intracellularly and extracellularly from UV damage, two projects were devised. For the intracellular experiment, all genotypes of Escherichia Coli are used. Overnight cultures were induced with arabinose and some genotypes were not induced with arabinose and incubated overnight at 30°C. After incubation, the optical spectrum of the cells was measured. After incubation, the optical density was measured at 600 nm and the right dilution was selected to achieve 100 colony-forming units on the plate. The plates were irradiated at different energies per area and incubated overnight at 37°C. The growth of the colonies on the plates were documented the next day.
To use Dsup to protect surfaces, the protein needs to work extracellulary. The protein was overexpressed and isolated from E.coli cells. The purified and concentrated protein was added to RPMI media and used during the experiment to cultivate the HeLa-cells. A live-cell imaging experiment was performed to find out if there are protective properties due to the Dsup. To compare the results, the experiment was performed with and without adding the Dsup as protective agent.

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# Team:Stuttgart/Experiments

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Projects

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[Protocols](https://2021.igem.org/Team%3AStuttgart/Experiments)
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Experiments and Protocols
Experiments and Protocols

# Methods

## Media and antibiotic

---

## LB Agar for *E.coli*

1. 10 g/L tryptone.

2. 10 g/L NaCl.

3. 5 g/L yeast extract.

4. 20 g/L agar.

5. Autoclave the bottle with medium.

6. Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

## LB Medium for *E.coli*

1. 10 g/L tryptone.

2. 10 g/L NaCl.

3. 5 g/L yeast extract.

4. Autoclave the bottle with medium.

5. Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

## TB-Medium

1. 12 g to 900 ml tryptone.

2. 5 g to 900 ml glycine.

3. 24 g to 900 ml yeast extract.

4. Autoclave bottle with medium.

5. Add 100 ml 10x TB-Salt

## TB salt

1. 23,1 g/L KH2PO4.

2. 125,4 g/L K2HPO4.

## Ampicillin solution

1. Prepare a solution containing a mass concentration of 150 mg/mL of ampicillin.

2. Dissolve ampicillin in dH20.

3. Filter the ampicillin solution through a syringe with a suitable filtration tip.

4. Aliquot the solution and store at -20 °C.

5. 1ml Ampicillin was added to 1 L medium.

---

## Cloning

---

## Processing of the ordered primers and fragments

1. Centrifuge the fragments and primers with Short Spin for approx. 10 sec.

2. Dilute the fragments and primers with ddH2O (final concentrations 50 nM for fragments and 100 µM for primers).

3. Vortex thoroughly

4. Short spin for approx. 10 sec.

## Primer and fragment sequences

Sequence of wildtype *dsup* fragment, optimized for *E.coli* :  [here is the sequence](https://static.igem.org/mediawiki/2021/0/0d/T--Stuttgart--WT_dsup_Ecoli2.txt)

Sequence of N-terminal *dsup* fragment, optimized for *E.coli* :  [here is the sequence](https://static.igem.org/mediawiki/2021/e/ee/T--Stuttgart--T_NTerm_dsup_E.coli.txt)

Sequence of C-terminal *dsup* fragment, optimized for *E.coli* :  [here is the sequence](https://static.igem.org/mediawiki/2021/1/12/T--Stuttgart--T_CTerm_dsup_Ecoli.txt)

Sequence of C-terminal *dsup* fragment, optimized for HeLa :  [here is the sequence](https://static.igem.org/mediawiki/2021/d/d0/T--Stuttgart--T_CTerm_dsup_HeLa.txt)

Sequence of wildtype *dsup* forward primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of wildtype *dsup* reverse primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of N-terminal *dsup* forward primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of N-terminal *dsup* reverse primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of C-terminal *dsup* forward primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of C-terminal *dsup* reverse primer, optimized for *E.coli* :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of N-terminal *dsup* forward primer, optimized for HeLa :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

Sequence of N-terminal *dsup* reverse primer, optimized for HeLa :  [here is the sequence](https://2021.igem.org/File%3AT--Stuttgart--T_Primer_2021_Stuttgart.docx)

## PCR for amplification

1.    pBAD18
and the *dsup* Fragments for *E. coli*

Method for 50 µl approach:

·       The
components listed in Table 1 were mixed in a PCR tube and run threw the cycles listed in Table 2.

Table 1: Reagents and corresponding concentrations used for a single PCR
amplification

| Components | Volume | Concentration |
| --- | --- | --- |
| Q5 2x Master Mix | 25 µl | - |
| 10 µM Primer fw | 2,5 µl | 0,5 µM |
| 10 µM Primer rv | 2,5 µl | 0,5 µM |
| ddH2O | 19 µl | - |
| DNA Template | 1 µl | - |

Table 2: General heat cycler program for PCR amplification. The cycle
steps were repeated for 30 times for the fragments and 40 times for the vector

| Step | Temp for fragment | Time for fragment | Temp for vector | Time for vector |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98°C | 30 s | 98°C | 30 s |
| Cycle: Denaturation | 98°C | 10 s | 98°C | 10 s |
| Cycle: Annealing | 70°C | 30 s | 70°C | 30 s |
| Cycle: Elongation | 72°C | 90 s | 72°C | 180 s |
| Final Extension | 70°C | 150 s | 70°C | 210 s |
| Hold | 8°C | - | 8°C | - |

2.    pcDNA3.1
and *dsup* fragments for HeLa

Method for
50 µl approach:

·       The
components listed in Table 3 were mixed in a PCR tube and run threw the cycles listed in Table 4.

Table 3: Reagents and corresponding concentrations used for a single PCR
amplification

| Components | Volume | Concentration |
| --- | --- | --- |
| Q5 2x Master Mix | 25 µl | - |
| 10 µM Primer fw | 2,5 µl | 0,5 µM |
| 10 µM Primer rv | 2,5 µl | 0,5 µM |
| ddH2O | 19 µl | - |
| DNA Template | 1 µl | - |

Table 4: General heat cycler program for PCR amplification. The cycle
steps were repeated for 30 times for the fragments and 40 times for the vector

| Step | Temp for fragment | Time for fragment | Temp for vector | Time for vector |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98°C | 30 s | 98°C | 30 s |
| Cycle: Denaturation | 98°C | 10 s | 98°C | 10 s |
| Cycle: Annealing | 71.5°C | 30 s | 72°C | 30 s |
| Cycle: Elongation | 74°C | 90 s | 74°C | 210 s |
| Final Extension | 71.5°C | 150 s | 72°C | 210 s |
| Hold | 8°C | - | 8°C | - |

## Preparation of the agarose gel

1. Briefly boil 0,8% w/v Agarose I n1x TAE Buffer until it´s solved.

2. Add 5 µl of Midori Green or 1 µl of Green Pack.

3. Pour into the mold and wait until the gel is fully polymerized.

4. Remove air bubbles or place in the bottom corner/side of the mold.

## Gelectrophoresis

1. Preparation of a 0,8% w/v agarose gel.

2. Add 5 µl or 10 µl loading dye (6x) to 25 µl or 50 µl of the PCR product, respectively.

3. Mix the mixture well using a pipette and load the whole volume in the gel pocket.

4. Run the gel on 100 V for 50 minutes.

5. Visualize your DNA fragments with UV light.

## DNA Gel Extraction

The DNA gel extraction was carried out with the Monarch® DNA Gel Extraction Kit from NEB. The gel extraction was performed as described in the protocol of this kit [(https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020).](https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020)

All centrifugation steps will be performed at 13,000 rpm.

1. In the end the DNA was eluted with 10 µl DNA Elution Buffer.

2. DNA concentration is determined by using a Nanodrop.

## PCR purification

The PCR purification was carried out with the Monarch® PCR and DNA Cleanup Kit from NEB. The PCR purification was performed as described in the protocol of this kit  [(https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol).](https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol)

All centrifugation steps will be performed at 13,000 rpm.

Dilute sample with DNA Cleanup Binding Buffer (from NEB Monarch® PCR and DNA Cleanup Kit), for dsDNA > 2 kb ratio Binding Buffer:Sample 2:1, for dsDNA > 2 kb ratio Binding Buffer:Sample 5:1.

## Gibson Assembly

1. In approach A: 3 µl fragment and 2 µl vector is added to 15 µl of Gibson Master Mix.

2. In approach B: 2,5 µl fragment and 2,5 µl vector is added to 15 µl of Gibson Master Mix.

3. In approach C: 2 fragment/1 vector mass ratio is added to 10 µl NEBuilder®.

4. For approach A and B the sample was incubated for 1 hour at 50 degrees.

5. For approach C the sample was incubated for 15 minutes at 50 degrees.

## DNA cleanup and concentration

The DNA cleanup and concentration was carried out with the Monarch® PCR and DNA Cleanup Kit from NEB. The DNA cleanup and concentration was performed as described in the protocol of this ki  [(https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol).](https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol)

All centrifugation steps will be performed at 13,000 rpm.

Dilute sample with DNA Cleanup Binding Buffer (from NEB Monarch® PCR and DNA Cleanup Kit), for dsDNA > 2 kb ratio Binding Buffer:Sample 2:1, for dsDNA > 2 kb ratio Binding Buffer:Sample 5:1.

## Preparation of *E.coli* Overnight Cultures

1. Add 5mL of LB or TB medium, depending on the purpose, to a culture tube.

2. Add 5 µL of the ampicilin.

3. Use a pipette tip to pick a single colony from the plate.

4. Immerse pipette tip in the medium-antibiotic mixture and incubate at 37°C.

## Competent cell production

The evening before, inoculate pre-culture in LB-medium with single colonies and incubate at 30°C. The next morning over-inoculate in 20 ml LB medium and incubate at 37°C until the OD600 value is about 0.4.

1. Centrifuge the culture at 13000 rpm for 6 min at 4°C.

2. Discard the supernatant and resuspend the pellet in 1 ml 0.1 M CaCl2 (ice cold).

3. Centrifuge the cells at 13000 rpm for 1 min 4°C.

4. resuspend the pellet in 0.2 ml 1 ml 0.1 M CaCl2.

## Transformation into *E. coli* cells

1. Thaw 25 µL of competent cells on ice.

2. Incubate for 30 min on ice.

3. Place the tube with the cells and DNA in thermocycler at 42 degrees for 45 sec.

4. Place the cells in ice for 1 min.

5. Add 450 µL of LB medium to the cells.

6. Plate out 100 μl of the cell suspension directly onto LB agar culture plates with the appropriate antibiotic.

7. Incubate the culture plates overnight at 37 °C.

For transformation of the empty vector, 1 µl of plasmid DNA was added to 25 µl of competent cells and for transformation of the Gibson plasmid, 3 µl was added. The Gibson plasmid was centrifuged at 13000 rpm for 1 minute before plating out in the agar plates, 350 µl of the supernatant was discarded, resuspended and the remainder was plated out onto the agar plates.

## Plasmid Isolation

The plasmid isolation was carried out with the innuPREP Plasmid Mini Kit 2.0from Jena Analytik. The plasmid isolation was performed as described in the protocol of this kit. [(https://www.analytik-jena.de/fileadmin/content/products/02\_Kits/innuPREP\_Plasmid\_Mini\_Kit\_2\_0/Manual\_innuPREP\_Plasmid\_Mini\_Kit\_2.0.pdf).](https://www.analytik-jena.de/fileadmin/content/products/02_Kits/innuPREP_Plasmid_Mini_Kit_2_0/Manual_innuPREP_Plasmid_Mini_Kit_2.0.pdf)

All centrifugation steps will be performed at 13,000 rpm.

## Protein expression

1. (Transform plasmid into BW25113. Plate on antibiotic selection plates and incubate overnight at 37°C.

2. Add a single colony to 5 ml TB medium still containing TB salt and ampilicin and incubate overnight at 37°C.

3. Inoculate the whole overnight culture into 500 ml of TB medium.

4. Incubate at 37°C with shaking.

5. Continuously measure OD at 600 nm and record.

6. When the OD value is between 0.4 and 0.6, induce with 5 ml of 2% arabinose to total volume.

7. Express protein at 37°C, at 30°C or at room temperature overnight.

## Protein purification: Nickel affinity chromatography

1. Equilibrate the HisTrap FF column with 5 column volume (CV) of binding buffer.
2. Apply the lysed cell extract.
a) C-term was purified on a 10 mL column.
b) N-term was purified on a 30 mL column.
3. Wash with 5 CV binding buffer.
4. Elute with 5 CV 500 mM imidazole in a one-step elution (used with C-term lysate) or a gradient (used with N-term lysate).
5. Collect the peak in a Falcon tube.
6. Pool the samples of the same variant.
7. Desalinate the samples on a HiTrap FF column, with Tris(Hydroxymethyl)Methylamin-Hydrochlorid (Tris-HCl) at pH 7.6.

## Bradford Assay

1. Prepare 1 mg/mL BSA standard solution by dissolving 1 mg Bovine Serum Albumine (BSA) in 1 mL ddH2O
2. Pipet the triplicate standard samples directly into cuvettes according to following scheme:

| BSA [µg] | BSA standard [µL] | H2O [µL] |
| --- | --- | --- |
| 0 | 0 | 100 |
| 2 | 2 | 98 |
| 5 | 5 | 95 |
| 7 | 7 | 93 |
| 10 | 10 | 90 |
| 15 | 15 | 85 |
| 20 | 20 | 80 |

3. Before adding the Bradford reagent make sure the photometer is set to 595 nm
4. Add the Bradford reagent and measure after exact 5 min (invert the samples by using Parafilm during the 5 min)
5. Note the absorbance at 595 nm.
Preparing samples:
1. Always use 100 µL of the diluted sample and add 900 µL of Bradford
2. Example: Use 2 µL of a 1:10 diluted sample with 98 ddH2O
3. Measure as in steps 3.-5. of the preparation of the calibration line
4. Adjust the used sample volume as needed to stay in the range of the standard samples.
Linear range: 2-25 µg/mL

## SDS-PAGE (qualitative)

1. Apply approximately 10 µg protein per sample, if needed dilute the sample.
2. Mix 7,5 µL of the samples with 7,5 µL 2x SDS loading buffer.
3. Boil at 95 °C for 10 min.
4. Apply 5 µL of marker and 15 µL of each sample mixed with loading buffer.
5. Run SDS-PAGE for 1,5 h at 100 V and 200 mA.
6. Stain with Coomassie for 40 min.
7. Destain with 20 % acetic acid for 60 min and subsequently with ddH2O overnight

## HeLa cultivation

Medium: Gibco Roswell Park Memorial Institute (RPMI) 1640 Medium + 10% FBS + 1% PenStrep.
Washing buffer: phosphate-buffered saline (PBS).
Detaching Reagent: Trypsin-EDTA.
Staining solution: Trypanblue.
All cell cultivation work was performed on a safety cabinet under aseptic conditions. Culture medium is stored in the fridge at 8 ° C, while trypsin has to be stored in the freezer at -20° C. The PBS was stored at RT. The culture media as well as the enzymes used were warmed to RT before use. The cultivation was carried out at 37 °C and 5% CO2. To cultivate the cells, cell culture flasks in 2 sizes were used (T75 and T25).

## Splitting

HeLa cells are adherent, as a result, the given space is limited. To ensure that the cells don’t overgrow each other, they have to split into several flasks to give them enough space.
1. Remove the medium.
2. Wash with PBS.
3. Add 2 ml Trypsin-EDTA (1x).
4. Incubate at 37 °C for 5 min (until cells are detached).
5. Transfer cells into a 10 ml falcon.
6. Centriuge at 2000 rpm for 5 min.
7. The pellet is resuspended in medium and then divided into new T75 flasks. If necessary the cells can be counted.

## Counting

For the live cell imaging experiments, as well as for the correct cultivation, the determination of the cell number is necessary. To count the cells, a Neubauer chamber and Trypanblue is used.
1. Detach the cells as described before.
2. Resuspend the cell pellet in 1 ml medium.
3. Take 20 μl of the cell suspension and add 80 μl Trypanblue.
4. Transfer 20 μl to the Neubauer chamber.
5. The number of cells is counted under a microscope.
6. The total number of cells (zz) is calculated by multiplication of the dilution factor, cell number, the voulum of the sample and the factor 10^4:
zz = counted cell number \* 5 \* 10^4 \* 1

# Experiments

## First irradiation Experiment with intracellular Dsup in *E. coli*

1. Apply approximately 10 µg protein per sample, if needed dilute the sample.
2. Mix 7,5 µL of the samples with 7,5 µL 2x SDS loading buffer.
3. Boil at 95 °C for 10 min.
4. Apply 5 µL of marker and 15 µL of each sample mixed with loading buffer.
5. Run SDS-PAGE for 1,5 h at 100 V and 200 mA.
6. Stain with Coomassie for 40 min.
7. Destain with 20 % acetic acid for 60 min and subsequently with ddH2O overnight

## Second irradiation experiment with *E.coli*

To eliminate the potential of a negative effect by the antibiotic on the cells, no ampicillin was used in the second experiment. There weren’t enough materials for a complete second experiment with triplicates for each genotype. Therefore, the same genotype with C-terminal His-Tagged Dsup as in the extracellular experiments was used, for better comparison. To minimize the variability introduced by the many dilution steps, the cells were only grown to a OD600 of 0,1. Please read the according paragraph in the troubleshooting section for further information about the difficulties of this experiment.
1. Inoculate 5 mL LB Medium with cells.
2. Add 50 µL 200 g/L Arabinose, except to the negative control.
3. A OD600 0,1 value was reached after approximately 3 h.
4. Dilute the suspension directly in 1:10 dilution steps to a total dilution of 10-5.
5. Plate 100 µL of the 10-5 diluted suspension on agar-LB-plates.
6. Incubate a 30 °C for 30 min.
7. Irradiate the cells at 100 µJ/cm2 without the cover of the agar plate.
8. Cover the plates again and place them back into the incubator.
9. Let the cells grow overnight at 30 °C.
10. Count the colonies on the next day.

## Live Cell Imaging

The Zeiss Axio Observer system with an integrated incubator was used for the live cell imaging experiments. The cells were incubated under standard conditions. In most live cell imaging experiments, the structures of the cell are stained. We would like to exclude the influence of a fluorophore on the protein, therefore the experiments were carried out without fluorophores. To achieve a realistic experimental setup, the cells were irradiated with a wavelength of 380 nm. Although there are only a few dyes that are excited at this wavelength (1-anilinonaphthalene-8-sulfonic acid), this range already belongs to UVA radiation. UVA is the lowest-energy form of UV radiation, but it can pass through the earth's atmosphere unhindered. [1]

For the live cell imaging experiments Hela cells were used. First, a setting had to be found which shows a phototoxic effect, but which does not immediately kill the cells. Such a setting would not correspond to a real experiment and no differences would occur between positive and negative control. Under these conditions, the DNA protection potential of intracellularly Dsup should be verified. Unfortionally, the Gibson assembly for the Hela plasmid did not work. Therefore, this part could not be performed. The other part is to test the extracellular Dsup protection potential. For this part, the purified Dsup is added to the media of the Hela cells.

[1] https://www.bfs.de/DE/themen/opt/uv/einfuehrung/einfuehrung\_node.html (20.10.2021)

## Experimental setup

1. Two times: Seed 30 000 HeLa cells to a confocal dish (20 mm)
   (one positive control without any treatment)
2. Transfer the dish into the incubator Axio Observer (37 °C and 5% CO2)
3. Choose settings by ZEN 3.1 (blue edition)
   Two blocks can be set up. The first block is identical for each test. The cells were only exposed to brightfield every 15 min for 20 h. They can recover from the transport to the microscope. The second block (approximate 52 h) determines phototoxicity. The setups for block two are shown in the following table.

![](https://static.igem.org/mediawiki/2021/5/5a/T--Stuttgart--TabelleHeLa.png)

For the extracellular experiment, the settings of test 3 were chosen. A quadripartite confocal dish was used to test different Dsup concentrations:

1. Seed 18 000 HeLa cells per quarter
2. Add different amounts of purified Dsup to the media (0 ng, 450 ng, 4500 ng, 0,1125 mg)
3. Choose settings of Test 3 by ZEN 3.1
4. Transfer the dish into the incubator Axio Observer (37 °C and 5% CO2)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stuttgart/Results

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Results
Results

# Results of the Escherichia Coli experiments

In the experimets, we performend, we used different variants of the Dsup protein: the wildtype protein (WT),N-term ( a His-Tag on the N-terminus),C-Term (a His-Tag on the C-terminus).

The vector and the fragments of the different Dsup variants are shown in Fig. 1. All Variants were successfully cloned into the pBAD18 vector.

![](https://static.igem.org/mediawiki/2021/d/db/T--Stuttgart--1.PNG)

Fig. 1: Agarose gelelectrophoresis of the fragment and vector used within the E. coli experiments. The Quick-Load 1 kb Plus DNA Ladder was used. M: Marker, V1/V2/V3: pBAD18 vector, F1: WT Dsup fragment, F2: C-term Dsup fragment, F3: N-term Dsup fragment.

## Transformation

The plasmids were transformed into cells of the effective transformation strain DH5α. Afterwards, they were isolated and transformed into the expression BW25113 strain.

## Expression

The transformed BW25113 cells were used to express Dsup at different temperatures. In the first run, the WT and N-term were cultivated at 37°c for 340 minutes (Fig. 2). The OD600 values were measured in regular intervals to determine the growth curve and optimal point of induction with arabinose. After 100 minutes the expression of both variants was induced with 5 mL arabinose. The process was determined at the stationary phase.

![](https://static.igem.org/mediawiki/2021/6/6b/T--Stuttgart--2.PNG)

Fig. 2: Growth curves of WT and N-term E. coli cultures. The process was started at 09:15 am and was stopped after 340 minutes. The expression of Dsup was induced after 100 minutes.

In the second run (Fig. 3), the C-term variant was cultivated over the span of 505 minutes. One culture was kept at room temperature, while the other was cultivated at 37°C like in the first run. The expression of Dsup was induced by the addition of 5 ml arabinose solution after 140 minutes. The second culture at 37 °C shows a stronger growth. The culture, incubated at room temperature, grows slower, but the protein folding might be less affected by the temperature.

![](https://static.igem.org/mediawiki/2021/b/b0/T--Stuttgart--3.PNG)

Fig. 3: Growth curves of C-term E. coli cultures at room temperature (RT) and at 37 °C. The process was started at 9:00 am and stopped after 505 minutes. Expression of Dsup was induced after 180 minutes.

The second expression of N-term was performed at 30 °C and the growth is shown in Fig. 4. The expression of the Dsup was induced after 180 min.

![](https://static.igem.org/mediawiki/2021/0/04/T--Stuttgart--5.png)

Fig. 4: Growth curves of two equal N-term Dsup E. coli cultures at 30°C. The process was stopped after 560 minutes. Expression of Dsup was induced after 180 minutes.

The harvested cells from the C-term expressions at different temperatures were pooled for the purification. The N-term samples of the different expressions were pooled as well. Then the cells were lysed.

## Purification and desalination

The purification of the C-term cell lysate was performed on a 10 mL HisTrap FF column according to the chromatogram in Fig. 5. The column was loaded with 10 mL as shown by the conductivity plateau. Then the His-Tag was eluted with imidazole in a one-step elution, resulting in three peaks. The first one at 15,5 mL wasn’t recognized by the software but was collected separately from the second peak at 16.22 mL. The collection of the second peak was stopped as soon as the conductivity started to rise at around 16 mL. The last peak at 18.34 mL wasn’t collected.

![](https://static.igem.org/mediawiki/2021/e/e7/T--Stuttgart--bild1.png)

Fig. 5: Purification chromatogram of the C-terminal His-Tagged Dsup

The desalination of the protein was performed by using affinity chromatography, using a HisTrap FF column
(Fig. 6). The solution of the second peak was collected until the conductivity started to rise at approximately 11 mL. The same procedure was used to desalt the solution of the first peak (purified C-term sample).

![](https://static.igem.org/mediawiki/2021/f/f0/T--Stuttgart--bild2.png)

Fig. 6: Desalination chromatogram of peak 2 of the purified C-terminal His-Tagged Dsup

For the purification of the N-term cell lysate 30 mL were applied to the column. After loading the solution on the column, the samples were eluted again, but with an imidazole gradient elution. Fig. 7 shows, that small concentrations of imidazole already elute the His-Tag of the N-term Dsup. The first peak at 104.34 mL and the second peak at 116.17 mL were collected separately.
![](https://static.igem.org/mediawiki/2021/2/2c/T--Stuttgart--bild3.png)

Fig. 7: Purification chromatogram of the N-terminal His-Tagged Dsup

In Fig. 8 the desalination shows several plateaus during the desalination of the second peak of the N-term purification. The desalted solution from peak 2 was collected in one Falcon tube until the conductivity started to rise at approximately 11 mL. For the sample of the first peak, the same procedure was performed and the solution was collected in a separate falcon tube.

![](https://static.igem.org/mediawiki/2021/7/79/T--Stuttgart--bild4.png)

Fig. 8: Desalination chromatogram of peak 2 of the purified C-terminal His-Tagged Dsup

## Bradford protein assay

To evaluate the samples from the different peaks of the purification step, the protein concentration was determined. The raw data and dilution factors of the samples measured with the Bradford protein assay are shown in Tab. 1. The coefficient of determination of the BSA calibration line in Fig. 9 is 0,9849.

![](https://static.igem.org/mediawiki/2021/5/5a/T--Stuttgart--bild5.png)

Fig. 9: BSA calibration line of the Bradford protein assay.

By converting the given equation 1 in Fig. 1 into equation 2 the protein concentration of the samples can be determined, whereas y stands for the absorbance and x defines the protein mass.

y=0,0281∙x+0,0341 (equation 1)

x=(y - 0,0341)/0,0281 (equation 2)

The mean absorption of the C-terminus was 0,37 and 20 µL of undiluted sample was used. The protein mass is determined in equation 3 and subsequently the concentration is determined in equation 4.

x=(0,37-0,0341)/0,0281=11,95 µg (equation 3)

concentration\_(C-terminus raw extract)=(11,95 µg)/(20 µL)=0,60 (µg)/(µL) (equation 4)

The highest protein concentration was found in the N-terminal His-Tagged cell lysate. The C-term peak 2 solution has the highest protein concentration of the purified and desalted samples with 0.54 mg/mL before and 0.45 mg/mL after sterile filtration.

Tab. 1: Bradford protein assay of the cell lysates and purified samples

![](https://static.igem.org/mediawiki/2021/3/33/T--Stuttgart--table1.png)

## SDS-Page

The SDS-PAGE Gels in Fig. 10 show all the samples from the expression of the Dsup protein of the different genotypes. The "pellets" are the cell pellets, which were generated at the end of the expression after the last centrifugation step.
After four hours a slight band at approximately 45 kDa is visible.

![](https://static.igem.org/mediawiki/2021/b/bd/T--Stuttgart--gele1.png)

Fig. 10: SDS-PAGE of all expression samples
The SDS-PAGE Gels include the samples of all genotypes at the beginning, before and after arabinose induction and at the end of the expressions carried out at RT, 30 °C and 37 °C. For a better overview, the time of the expression or whether it’s a pellet is written below the sample names. M: Marker, C1: C-term 37 °C 0 h, C2: C-term RT 0 h, C3: C-term 37 °C 4,25 h, C4: C-term 37 °C pellet, C5: C-term RT 4,75 h, W1: WT 30 °C before arabinose 2 h, W2: WT 30 °C 4 h, W3: WT 30 °C pellet, N1: N-term 30 °C before arabinose induction 2 h, N2: N-term 30 °C 4 h, N3: N-term 30 °C pellet, N4: N-term RT 7 h, C6: C-term 30 °C 5 h, C7: C-term RT pellet, W4: WT RT pellet, N5: N-term RT pellet, C8: C-term RT pellet, C9: C-term 30 °C 0 h, C10: C-term 30 °C 4h, W5: WT RT 0 h, W6: WT RT 7 h, N6: N-term RT 0 h.

In Fig. 11 and Fig. 12 the cell lysates and the desalted samples are shown. The additional samples of the expression in Fig. 11 can be compared to the lysed and purified samples. After 5 h of C-term expression, multiple bands can be seen across the size standard, whereas only two bands at approximately 38 kDa are seen in the pellet. Only very weak bands are visible in the sample of the cell lysate. The protein concentration of the C-term lysate was first determined for the sample preparation for the SDS-PAGE in Fig. 12. A lesser volume of the C-term lysate as needed was applied. But the bands for the maximum volume of undiluted 7.5 µL C-term cell lysate in Fig. 12 are even weaker than in Fig. 11. There are no visible bands for the C-term peak 2 samples. The same result is shown in Fig. 12 for the other C-term peak and sterile filtered sample. At the beginning of the N-term expression in RT, only one weak band at approximately 40 kDa is visible, whereas the N-term cell lysate sample shows several bands with one outstanding band at 43 kDa. The purified N-term samples of all peaks show a clear band at approximately 43 kDa and a second weaker band at around 38 kDa. Except for peak 1 of expression 1, where the bands are less present. In Fig. 8 also the N-term peak 2 of expression 2 shows a clear band at 43 kDa as the other desalted N-term samples.

![](https://static.igem.org/mediawiki/2021/6/6d/T--Stuttgart--gele2.png)

Fig. 11: Fig. 11: SDS-PAGE of C-term and N-term expression and the purified samples
M: Marker, C1: Expression C-term 30 °C 0 h, C2: C-term 30 °C 5 h, C3: C-term pellet RT, C4: C-term cell lysate, C5: C-term peak 2, N1: Expression N-term RT 0 h, N2: N-term cell lysate, N3: N-term peak 1 expression 1, N4: N-term peak 1 expression 2, N5: N-term peak 2 expression 2

The C-term sample from the beginning of the expression in Fig. 12 shows weak bands at approximately 35 and 37 kDa. As mentioned earlier the bands of the C-term cell lysate in Fig. 8 are nearly not visible. The C-term peak 1 sample in Fig. 12 leaked between the gel and the glass plates of the gel.

![](https://static.igem.org/mediawiki/2021/5/54/T--Stuttgart--gele3.png)

Fig. 12: SDS PAGE of all purified samples
M: Marker, C1: Expression 30 °C 0 h, C2: C-term cell lysate, C3: C-term peak 1, C4: C-term peak 2, C5: C-term peak 2 sterile filtered, N1: N-term cell lysate, N2: N-term peak 1 expression 1, N3: N-term peak 2 expression 1, N4: N-term peak 1 expression 2, N5: N-term peak 2 expression 2.

## Experiment

In the first irradiation experiment the Dsup expression was induced in all genotypes except in the WT n (non-induced) as a negative control. The different E. coli genotypes were irradiated with different energy levels as seen in Fig. 13 and Tab. 2. There were less than 100 colonies on each plate, even in the uniradiated controls. A clear effect of the radiation is seen at radiation levels of 2500 µl/cm2. None of the genotypes survived a irradiation of 10000 µJ/cm2. The number of colonies varies between the plates, to make predictions of a trend, more replicates should have been performed.

![](https://static.igem.org/mediawiki/2021/5/5e/T--Stuttgart--Fig13.png)

Fig. 13: Irradiation of all genotypes at different energy levels.

Tab. 2: Raw data of the first irradiation experiment with different energy levels of all genotypes. As negative control cells without the induction of the WT Dsup expression were used: WT n: WT non-induced, WT: induced WT.

![](https://static.igem.org/mediawiki/2021/a/a8/T--Stuttgart--Tab2.png)

Therefore the experiment was performed with one genotype using a specific condition, with replicates (Fig. 14). The number of colonies varies between the plates. The radiation seems to have no effects on the induced C-term cells, whereas the number of colonies in the non-induced C-term genotype seems to decrease. As seen in the raw data of the second irradiation experiment, the total number of colonies is too low and the variation is high, so further experiments would be necessary to prove the protective effect of the Dsup.

![](https://static.igem.org/mediawiki/2021/0/09/T--Stuttgart--Fig.15.png)

Fig. 14: Second irradiation experiment of the C-term genotype

Tab. 3: Raw data of the second irradiation experiment of the C-term genotype. As negative control cells without the induction of the C-terminal His-tagged Dsup expression were used: C-term n: non-induced C-term, C-term i: induced C-term.

![](https://static.igem.org/mediawiki/2021/f/fd/T--Stuttgart--Tab.4.png)

## Troubleshooting

E. coli irradiation with intracellular expressed Dsup:

• Dilution: many dilution steps are susceptible for mistakes, which leads to amounts of colonies, which are not countable.

• Contamination:
We wanted to eliminate the possible effects of antibiotics on cell growth, therefore we didn’t use any antibiotics. But we couldn’t place the UV radiator under the clean bench so we had several contaminations at the first trial (data not shown). The set-up should be performed under clean and sterile conditions to avoid contaminations.

## Discussion

The cell growth of the expressions worked for all genotypes. The band, which can be found at approximately 43 kDa, matches with the given size of the native Dsup protein of 42.8 kDa [1]. Unfortunately, there could not be detected proteins by the SDS-PAGEs of the purified and desalted C-term Dsup, even though the highest protein concentration was measured in the C-term peak 2 samples. One reason for the missing Dsup bands could be the high protein concentration, which was detected in an elution step. When the samples are boiled before loading on the SDS-PAGE the imidazole remnants could lead to a hydrolyzation of the proteins [2]. The protein content in the C-term cell lysate seemed to decrease. The protein concentration of the C-term lysate was determined to right before the preparation of the second SDS-Page.
For the preparation of the first SDS-PAGE with desalted samples, it was assumed that the C-term cell lysate has a similar protein concentration as the N-term lysate, a smaller volume was used. In Fig. 10 the maximum volume of 7.5 µL of the C-term cell lysate was used but there are even fewer bands visible than in Fig. 9. It can be assumed that the C-term samples were exposed to room temperature for too long and proteases in the cell lysate degraded some of the proteins in this sample [3]. The total loss of protein bands in the C-term cell lysate rather indicates, that the sample preparation was incorrect, than no Dsup was expressed.
The high protein concentration and the high specificity of the HisTrap column contribute to the successful expression and purification. However, it cannot be ensured assuredly that the C-term Dsup protein was expressed.

The extracellular irradiation experiment showed that a high variation of the numbers of colonies occur. Most likely because of the possible mistakes, that can happen during the dilution steps. Also, it seems critical to avoid leftover suspension in the pipet tip, because there also was a high variety of colonies from the same dilution. The experiment needs to be repeated for all the genotypes with more replicates to achieve a representative result. Nevertheless, the critical irradiation energy at 2500 µJ/cm2 and a lethal energy level to all genotypes at 10000 µJ/cm2 could be observed.

[1] Chavez, C., Cruz-Becerra, G., Fei, J., Kassavetis, G. A., & Kadonaga, J. T. (2019). The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. ELife, 8, e47682. [(https://doi.org/10.7554/eLife.47682)](https://doi.org/10.7554/eLife.47682)

[2] Rapley, R. (2000). The Nucleic Acid Protocols Handbook. Springer Science & Business Media.

[3] Ali M, Suzuki H, Fukuba T, Jiang X, Nakano H, Yamane T. Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant. J Biosci Bioeng. 2005 Feb;99(2):181-6. doi: 10.1263/jbb.99.181. PMID: 16233776.

# Results HeLa live cell imaging

In the pre-experiment, three tests were performed to find the best adjustments to observe the test. In these videos two blocks were performed: the first block takes place without any radiation. In the second block, the cells were stressed with rays every 15 minutes, to find out their tolerance level for phototoxicity.

In the first test (test 1: 380 nm, 7s every 15 min), all HeLa cells died after a few hours. In the following test (test 2: 380 nm, 7s every 15 min), a filter was used that allowed only 10% of the rays to pass through. Even after 72 hours, no change in proliferation was visible. The results of test 3 (380 nm, 7s every 15 min) are shown in the following two video clips.

[

](https://static.igem.org/mediawiki/2021/0/00/T--Stuttgart--VideoTest.mp4)

[

](https://static.igem.org/mediawiki/2021/9/96/T--Stuttgart--VideoTest2.mp4)

The first video shows the first block. The cells are not stressed and proliferate constantly. The second video shows the second block of the third test. After approximate 11 hours, blebbing occurs. Probably the cells are dying due to the phototoxicity.
<p>

## Live cell imaging with extracellular Dsup protein

The actual experiment only shows block two, to find out, whether the Dsup has any impact. Due to time reasons, the experiment was only performed once. To test the extracellular Dsup protection potential, the protein with a His-tag at the C-terminus was used. Four different Dsup amounts (0 ng, 450 ng, 4500 ng, 0,1125 mg) were tested at the same time in a quartered confocal dish. Each quarter contains 18.000 HeLa cells.
The videos below show the third test with the addition of Dsup. The rays lead to the death of the cells even with a low exposure time. Due to the results of the pre-experiments, the cells were observed with the addition of DSUP within the media. By taking a look at the positive control, no dying of the cells can be observed. The cells with an addition of 450 ng and 4500 ng of Dsup don’t show any difference in their behaviour. Only the highest concentration (0,1125 mg) of Dsup leads to apoptosis of the cells

[

](https://static.igem.org/mediawiki/2021/5/53/T--Stuttgart--Dsup0.mp4)

[

](https://static.igem.org/mediawiki/2021/9/93/T--Stuttgart--Dsup1.mp4)

[

](https://static.igem.org/mediawiki/2021/4/47/T--Stuttgart--Dsup10.mp4)
[

](https://static.igem.org/mediawiki/2021/1/17/T--Stuttgart--Dsup250.mp4)

## Discussion

In the preliminary test with the untreated HeLa cells, we were able to show that they react sensitively to UVA radiation. Already 3s of 380 nm every 15 min led to the death of the cells after a few hours.
We could not prove a protective effect of the Dsup protein in this setup. However, this may have several reasons. Firstly, the preliminary experiment was carried out six weeks before the experiment with Dsup. The cells were therefore in different passage numbers. This could have a greater influence on the sensitivity of the cells than we thought. Secondly, the amount of protein might not have been high enough. Furthermore, only the C-term Dsup was tested. Possibly the His-tag has a negative influence on the folding of the protein, which is why it cannot show its potential. This assumption could have been refuted with the same procedure for the N-term. Unfortunately, there was not enough time for this. But we could show that the Dsup extracellularly has no negative influence on the proliferation of HeLa cells. The highest concentration of Dsup leads to the death of the cells, this is not the result, we have expected. But since the pre-experiment in this set-up hasn’t worked out as well and the cells were not in a good place already in the beginning, there can’t be made a clear prediction out of this result. The experiments have to be repeated to find out if Dsup has a positive impact.