iGEM 2021 — Team Hamburg

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# Notebook

[##### Cyanobacteria lab book 2021](https://static.igem.org/mediawiki/2021/a/ae/T--Hamburg--Cyano-lab-book.pdf)
[##### May 2021](https://static.igem.org/mediawiki/2021/8/84/T--Hamburg--Mai.pdf)
[##### June 2021](https://static.igem.org/mediawiki/2021/9/9b/T--Hamburg--Juni.pdf)
[##### July 2021](https://static.igem.org/mediawiki/2021/f/f3/T--Hamburg--Juli-lab-book.pdf)
[##### August 2021](https://static.igem.org/mediawiki/2021/1/1f/T--Hamburg--August.pdf)
[##### September 2021](https://static.igem.org/mediawiki/2021/7/7c/T--Hamburg--September.pdf)
[##### October 2021](https://static.igem.org/mediawiki/2021/a/a4/T--Hamburg--Oktober.pdf)

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# Team:Hamburg/Engineering

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# Engineering

### Designing flexible cloning cassettes for CYP enzymes

Modularity in construct design is important to allow the simple exchange of single parts for flexible assemblies of different related constructs. Particularly, for the design of our fusion protein it was essential to enable the exchange of cytochrome P450 and reductase enzymes, e.g. to analyse the effect of the linker sequence.

**Level 1:**
Initially we designed our parts to be assembled by golden gate assembly for each construct (e.g. fusion protein expression cassette) individually. After assembling the first constructs we realised that to test our system we needed to exchange parts of the sequence. This was not really possible with our construct design and we had to assembly the whole construct again. Which made it difficult to exchange parts of the construct like promoters. Therefore we aimed to improve the modularity of our construct design.

**Level 2:**
In a first step to allow more variability and easier combination of cytochrome P450 enzymes and reductases, restriction enzymes sites for MfeI (CAATG) and BamHI (GGATCC) were introduced at the N-terminus and C-Terminus of the cytochrome P450 enzymes and reductases respectively. Linkers were then created by annealing DNA oligos with AATG and GATC overhangs. Flexible linkers mainly containing glycine and rigid linkers mainly containing proline of different length were created. Introducing the MfeI and BamHI restriction sites to the construct leads to restriction in the flexibility of the linker sequence, since not all amino acids can be chosen freely. Two amino acids at the N-Terminus (Glu + Leu)) and two amino acids at the C-terminus (Gly + Ser) were fixed.
This new design improved the ability of our system in creating fusion constructs but all our other parts were still assembled individually. There was no common assembly syntax specified so for each assembly new primers needed to be obtained.
![](https://static.igem.org/mediawiki/2021/3/3a/T--Hamburg--parts_overview_LinkerV01.png)

Fig. 1: Linker design V 1.0. Expression cassette, MfeI and BamHI restriction sites

**Level 3:**
To even improve the usefulness of our parts, we then aimed to make our parts compatible with the MoClo standard of golden gate based IIS restriction enzyme assembly.
Thereby we expanded the *Common Genetic Syntax* for fusion sites to allow the creation of a) fusion proteins connected by linker sequences and b) multiple CDS expressed in an operon. All parts created according to this syntax are compatible with other MoClo collections like the Marburg collection. This advancement of the

![](https://static.igem.org/mediawiki/2021/7/75/T--Hamburg--parts_overview_MolClo.png)

Fig. 2: Common fusion site assembly syntax for our part design.

### Designing a CYP1A1-Activity Assay

One of our projects’ goals was building a flexible platform for the development of CYP/CPR-fusion proteins. This requires a cheap, fast and reliable assay to assess the functionality of different fusion constructs. Since the CYP enzymes we used in our project require expensive substrates and produce difficult to detect products they are not suitable for this purpose and we needed to select a model enzyme. As a model enzyme we chose CYP1A1 which catalyzes the dealkylation of 7-ethoxycoumarin, producing 7-hydroxycoumarin (fig. 3). This substrate is an ideal model, as it is readily available and can be tracked by fluorescence measurements.
![](https://static.igem.org/mediawiki/2021/5/59/T--Hamburg--eng-1.png)

Fig. 3: Convertion of 7-Ethoxycoumarine to 7-Hydroxycoumarine

Before testing our CYP1A1 constructs we first verified how well we could qualitatively and quantitatively measure both 7-ethoxycoumarin and 7-hydroxycoumarin. We started by measuring a dilution series of 7-ethoxycoumarin and 7-hydroxycoumarin in water, using wavelengths indicated in the literature [1] (exc: 390 nm; emi: 465nm) but observed no fluorescence. For our next test cycle we instead used the excitation wavelength (exc: 323 nm) indicated by the supplier [2] and used the plate-readers’ scanning mode to determine the [emission spectra](https://2021.igem.org/Team%3AHamburg/Results) of 7-ethoxycoumarin and 7-hydroxycoumarin. The emission maxima from the emission spectra were then used for further assay development.
With our completed CYP1A1 control construct (BBa-K3846369) we looked to iteratively optimize our assay through multiple design-build-test cycles. Thanks to our opentrons pipetting robot we were able to combine these design-build-test cycles and perform them in parallel in 96-well plates. We investigated different starting concentrations of 7-ethoxycoumarin, different dilutions of the cell-suspension and different cell-lines. While using wild type *E. coli* as a negative control and cell-suspensions diluted with water as a blank. Our experiments showed rapid conversion of the substrates in both *E. coli* transformed with our constructs and wild-type *E. coli*. No differences in the conversion rate could be detected.

These results show that even after our optimization cycles the assay still does not achieve the required reliability. Clearly the bacteria can already metabolize 7-ethoxycoumarin on their own, though one could expect that additional enzymes with catalytic activity towards 7-ethoxycoumarin would result in a measurable increase in the conversion rates. There remain two possible explanations for the results. Either our constructs are non-functional, possibly due to misfolding during expression or low expression concentrations. Alternatively our constructs are functional but the assays’ signal-to-noise ratio is too low to resolve the difference in activity.

To eliminate issues with the assays’ signal-to-noise ratio, an in vitro assay using purified protein is the logical next step. For this purpose we added 6xHis-tags to our proteins, but so far were unable to obtain sufficient quantities of the purified proteins for the next design-build-test cycle.

### Quantifying Terpenoid production

After optimizing our expression platform using CYP1A1 as a model substrate we will still need to verify if our actual terpene oxidizing CYP P450 enzymes work as expected and then quantify their output. As a measurement technique we settled on gas chromatography coupled mass spectrometry. This technique enables seperation of the terpenoid from complex metabolite mixtures and also enables accurate identification and quantification. For their identification, terpenoid spectra can be compared to spectra of authentic samples, which can either be obtained from the literature or measured on our own device. For the quantification gas chromatography peaks can be identified and integrated. These integrals can then be compared to the integrals of genuine analytical standards with known quantities.

### Parallel approach to testing fusion constructs

There are two key aspects to consider when engineering novel metabolic pathways in microorganisms. First, the DNA sequences of the enzymes need to be considered. Second, the expression conditions need to be optimized.

When optimizing the DNA sequences of enzymatic fusion proteins the assembly, cloning and subsequent expression and purification can pose significant bottlenecks when working with sequential design-build-test cycles. Therefore we decided to take a partial parallel approach to planning design-build-test cycles. We designed a library of each of our CYP enzymes with their native CPR (if available), the CPR from *A. thaliana* and the BM3R domain, both as fusion constructs and as individually expressed constructs on one plasmid. Sadly we did not successfully implement the production of terpenoid backbones and therefore could not test the designs we created. When taking this project further towards a real world application there are additional parameters that could be included in the library design. Different linker designs and different truncation patterns of the CYP and CPR enzymes could be used. Due to the exponential growth of library size when considering multiple parameters these were not considered while pursuing a proof of concept in this project.

For each CYP-CPR fusion construct individual expression conditions need to be determined, while the linkers and truncation patterns should have little impact on the optimal expression conditions. The expression conditions are dependent on several factors which can be optimized in sequential screening cycles. The following conditions should be investigated: inducer concentration, optical density at induction, induction time before cell harvesting and temperature during protein expression. For each of these parameters multiple different conditions should be investigated and both protein concentration and product titer should be determined. After selecting the optimal condition it should be used while screening for the next parameter.

[1] C. Manjon, B. J. Troczka, M. Zaworra, K. Beadle, E. Randall, G. Hertlein, K. S. Singh, C. T. Zimmer, R. A. Homem, B. Lueke, R. Reid, L. Kor, M. Kohler, J. Benting, M. S. Williamson, T. G. E. Davies, L. M. Field, C. Bass, R. Nauen, Curr. Biol. 2018, 28, 1137-1143.
[2] Sigma-Aldrich, can be found under https://www.sigmaaldrich.com/DE/de/product/sigma/e1379.

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# Team:Hamburg/Experiments

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# Experiments

### Agarosegel (1%) :

0.6 g/1.5 g Agarose

60 mL/ 150 mL TEA-Buffer heat in microwave until agarose is

dissolved. Cool down and add

6 μL/ 15 μL EtBr Transfer into gel slide and cool for 30 min.

### Electrophoresis:

45 min @120V

### Antibiotic Stocks:

34 mg/mL preparation of Chloramphenicol Stock (EtOH) (Cam: 34 mg/mL in Ethanol; Amp:

100 mg/mL in H2O; Kan: 50 mg/mL in H2O)

1. dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol

2. filter through a 0.22 μL filter to sterilize

3. use at 1:1000 dilution in LB or LB-agar

4. mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics

5. store at -20 °C

### Competent cells:

1. Grow the cells at 37°C till OD=0.2/0.4

2. Chill the culture on ice for 5 min.

3. Collect cells by centrifugation, 6000rpm 10min 4°

4. Gently resuspend the cells from 500ml LB in 40 mL cold Ca/glycerol buffer on

ice.

5. Incubate the cells on ice for 5~30 min.

6. Repeat step 3 and 4.

7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The

longer, the better)

8. Collect cells by centrifugation, 6000 rpm 10 min 4°C

9. Resuspend cells from 500ml LB in 6ml Ca/glycerol buffer.

10. Aliquote 150ul/tube. Freeze in liquid N2 and store in 80°C.

### Cryo Stocks:

1. Grow the Cells at 37°C till OD=0.2-0.4

2. Chill the culture on ice for 5 min.

3. Select cells by centrifugation, 6000 rpm/10 min/4°C

4. Gently resuspend the cells from 500ml LB in 40 mL cold Ca/glycerol buffer on

ice.

5. Incubate the cells on ice for 5~30 min.

6. Repeat stepp 3 and 4.

7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min.

(The longer, the better)

8. Collect cells by centrifugation, 6000 rpm for 10 min and 4°C.

9. Resuspend cells in 6ml Ca/glycerol buffer.

10. Aliquote 150μl/tube. Freeze in liquid N2 and store at -80°C.

### Extraction of Terpenoids:

We used the following methods to extract our products out of the reaction mixture.

**Method 1: Extraction from Cell Culture**

1. Mechanical lysis (optional)

2. Pick 100 µL of Lysate

3. Centrifugation for 3 min at 13300 g

4. Extract 3 times with 100 µL Ethyl Acetate

5. Dry the organic layer with NaSO4 and purificate it via filtering

6. 30 µL of the organic layer is diluted with 970 µL Ethyl Acetate

7. Sample is analysed by GC-MS

**Method 2: Extractive Fermentation**

1. After induction the cell culture is covered with a layer of dodecane

2. 100 µL of the dodecane layer is picked

3. Centrifugation for 3 min at 13300 g

4. 10 µL of the organic layer is diluted with 990 µL

5. Sample is analysed by GC-MS

### Gelextraktion:

1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade.

Cut a close to the DNA as possible to minimize the gel volume. Place the gel

slice into a pre-weighed 1.5 mL tube and weight. Record the weight of the gel

slice.

2. Add 1:1 volume of binding buffer to the gel slice (volume:weight).

3. Incubate the gel mixture 50-60 °C for 10 min or until the gel slice is completely

dissolved. Mix the tube by inversion every few minutes to facilitate the melting

process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly

before loading on the column. Check the color of the solution. A yellow color

indicates an optimal pH for DNA binding. If the color of the solution is orange or

violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the

mix will become yellow.

4. For less than 500 p or more than 10 kb add.

5. Transfer up to 800 μL of the solution to the GeneJET purification column.

Centrifuge for 1 minute. back into the same collection tube. Discard the flow-

through and place the column back into the same collection tube. Incubate again

10 minutes with 50 °C .

6. Add 700 μL of wash buffer to the GeneJET purification column. Centrifuge for 1

min. Discard the flow-through and place the column back into the same collection

tube. Repeat three times.

7. Centrifuge the empty GeneJET purification column for an additional 1 min to

completely remove residual wash buffer.

Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge

tube (not included). Add 10 bidest. H2O ( 50°C) and incubate for 10 minutes. And

centrifuge for one minute. Repeat once.

### Golden Gate Assembly

1. Mix together:

0.5 μL T4-DNA Ligase

2 μL of 10\* Ligase Buffer

0.5 μL Type IIS restriction Enzyme

100 ng Vector

equimolar amount of inserts

20 μL water QS.

Mix gently and place the tube on a thermocycler.

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time |
| Activation of the restriction enzyme | (15-120 cycles) 37°C | 5 min |
| Activation of the Ligase | (15-120 cycles) 16°C | 5 min |
| Inactivation of the enzyme | 55°C | 15 min |
| Inactivation of the Ligase | 85°C | 20 min |
| Hold | 4 °C | ꝏ |

### Miniprep (Thermo Scientific GeneJET Plasmid Miniprep KitK0502, K0503) :

1. Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL

of LB medium supplemented with the appropriate selection antibiotic. Incubate

for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask

with a volume of least 4 times the culture volume. Harvest the bacterial culture

by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room

temperature. Decant the supernatant and remove all remaining medium.

2. Resuspend cell lyses and neutralisation:

1. 250 μL resuspension solution and vortex
- 250 μL lysis solution and invert 4-6 times
- 350 μL Neutralization Solution and mix immediately and

thoroughly by inverting the tube 4-6 times. Centrifuge for 5 minutes

3. Binde DNA Transfer the supernatant to the Thermo scientific GeneJET Spin

Column and centrifugate for 1 minute.

4. Wash the colum with 500 μL wash solution and centrifugate for 30-60

seconds. Discard the flow-through again.

5. Centrifuge the empty column for 1 min.

6. Eluate purified DNA

7. Transfer the column into a new tube and add 50 μL Eluation buffer to the

column and incubate for 2 min.

### PCR

**PCR-Mix:**

|  |  |
| --- | --- |
| Volume [µL] | Ingredients |
| 25 | Mastermix |
| 2,5 | Forward Primer |
| 2,5 | Reverse Primer |
| 1 | DMSO |
| 18,5 | H2O |
| 0,5 | Polymerase (Q5, Phusion, S7 Fusion) |

**Program:**

|  |  |  |
| --- | --- | --- |
| 98°C | 30s |  |
| 98°C | 15s | | |
| 50-65°C | 20s | | 25x |
| 72°C | 90s | | |
| 72°C | 5min |  |
| 4°C | ∞ |  |

### Colony-PCR

**Colony-PCR-Mix:**

|  |  |
| --- | --- |
| Volume [µL] | Ingredients |
| 7,75 | H2O |
| 1 | DreamTag Buffer |
| 0,2 | dNTP’s |
| 0,5 | Primer VR |
| 0,5 | Primer VF2 |
| 0,05 | DreamTag |

**Program:**

|  |  |  |
| --- | --- | --- |
| 95°C | 10min |  |
| 95°C | 30s | | |
| 52°C | 30s | | 20x |
| 72°C | 4:15min | | |
| 72°C | 10min |  |
| 4°C | ∞ |  |

### Platereader

The Platereader assays were performed on a TECAN SPARK microplatereader

Excitation wavelength: **325 nm**

Emission wavelength: **455 nm**

The assay was loaded as followed:

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Stocksolution (S): 125 mM Ethoxy-Cumarine in EtOH | | | Cell types | BL21 | RIPL | C43 | WT |
| Stocksolution 2 (S2): 125 mM Hydroxy-Cumarine in EtOH | | |  |  |  |  |  |
|  |  |  |  |  |  |  |  |
| **Cell extract (Z)** | | (1:20) | (1:100) | (1:20) | (1:100) |  |  |
| **Ethoxy-Cumarine** | | *1* | *2* | *3* | *4* | *5* | *6* |
| 1.25 mM | *A* | 1 µL S+ 5 µL Z+ 94 µL H2O | 1µL S + 1µL Z + 98µL H2O | 1 µL S+ 5 µL Z+ 94 µL H2O | 1µL S + 1µL Z + 98µL H2O |  | 1µL S + 99 µL H2O |
| 0.125 mM | *B* | 10 µL S (1:100) + 5 µL + 85 µL H2O | 10µL S (1:100)+ 1µL Z + 89µL H2O | 10 µL S (1:100) + 5 µL + 85 µL H2O | 10µL S (1:100)+ 1µL Z + 89µL H2O |  | 10 µL S (1:100) + 90 µL H2O |
| 0.0125 mM | *C* | 1 µL S (1:100) + 5 µL + 94 µL H2O | 1µL S (1:100)+ 1µL Z + 98µL H2O | 1 µL S (1:100) + 5 µL + 94 µL H2O | 1µL S (1:100)+ 1µL Z + 98µL H2O |  | 1 µL S (1:100) + 99 µL H2O |
| 0.00125 mM | *D* | 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O | 0,1µL S (1:100)+ 1µL Z + 98,9µL H2O | 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O | 0,1µL S (1:100)+ 1µL Z + 98,9 µL H2O |  | 0,1 µL S (1:100) 99,9 µL H2O |
| 1.25 mM | *E* | 1 µL S+ 5 µL Z+ 94 µL H2O | 1µL S + 1µL Z + 98µL H2O | 1 µL S+ 5 µL Z+ 94 µL H2O | 1µL S + 1µL Z + 98µL H2O |  | 1µL S2 + 99 µL H2O |
| 0.125 mM | *F* | 10 µL S (1:100) + 5 µL + 85 µL H2O | 10µL S (1:100)+ 1µL Z + 89µL H2O | 10 µL S (1:100) + 5 µL + 85 µL H2O | 10µL S (1:100)+ 1µL Z + 89µL H2O |  | 10 µL S2 (1:100) + 90 µL H2O |
| 0.0125 mM | *G* | 1 µL S (1:100) + 5 µL + 94 µL H2O | 1µL S (1:100)+ 1µL Z + 98µL H2O | 1 µL S (1:100) + 5 µL + 94 µL H2O | 1µL S (1:100)+ 1µL Z + 98µL H2O |  | 1 µL S2 (1:100) + 99 µL H2O |
| 0.00125 mM | *H* | 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O | 0,1µL S (1:100)+ 1µL Z + 98,9µL H2O | 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O | 0,1µL S (1:100)+ 1µL Z + 98,9 µL H2O |  | 0,1 µL S2 (1:100) 99,9 µL H2O |

Protocol optimised to express mammalian proteins inside of Rosetta E. coli - a strain of E. coli containing plasmid encoding tRNAs carrying codons that are rare inside of prokaryotic cells. This protocol has been adapted from one sent by Xiang.

Steps used here describe the use of purification through a 6x-His-SUMO tag, but other purification tags can be used, though buffers may need to be optimised.

Protocol assumes the use of kanamycin, but this as well should be changed depending on plasmid selection gene used.

**Buffers to prepare:**

The pH-Value has to be adjusted to the pI-Value of the Proteine.
**Binding buffer** (1 L):

* 20 mM HEPES-NaOH pH 7.4 (20 mL of 1 M)
* 500 mM NaCl (100 mL of 5 M)
* 10 mM Imidazole (10 mL of 1 M)

**Washing buffer** (500 mL)

* 20 mM HEPES-NaOH pH 8.0 (10 mL of 1M)
* 500 mM NaCl (50 mL of 5 M)
* 20 mM Imidazole (20 mM Imidazole)
* Add Glycerol to 10% in final volume

**Elution buffer** (500 mL)

* 20 mM HEPES-NaOH pH 8.0
* 500 mM NaCl
* 400 mM Imidazole
* Add Glycerol to 20% in final volume

**Storage buffer** (100 mL)

* 20 mM HEPES-NaOH pH 8.0
* 150 mM NaCl

All Buffers are filter sterilised and should be pre-chilled at 4°C prior to use.

**Other Reagents:**

* Rosetta E. coli competent cells
* Appropriate selection antibiotic (kanamycin, etc...) - filter sterilized
* Ni-NTA Resin (can be different depending on specific purification tag used)
* 1 M IPTG - Filter sterilised
* LB media – autoclaved
* (optional) 1 M MgCl2, 200 mM CaCl2, DNase

### Protein Expression 1. Transform Rosetta with plasmid, and streak onto appropriate selection plates. Incubate overnight at 37°C until colonies are easy to pick up, avoiding formation of satellites. 2. Add 50 μL of kanamycin stock to 50 mL of LB media (50 μg/mL). Inoculate media with a single colony from step 1. Incubate overnight at 37°C at 250 RPM. 3. Into 1 L LB media, add kanamycin stock to 50 μg/mL, and ethanol to 2% of final volume. Inoculate 1 L media with start culture. Incubate at 37°C at 200 RPM until OD600 reaches 0.6-0.8 (Roughly 2-3 hours depending on confluence of start culture). 1. Expression culture can be scaled up or down, depending on the quantity of protein required. 2. Ethanol slightly hinders growth but also reduces formation of inclusion bodies. Once at desired OD, add IPTG to a final concentration of 0.7 mM to induce protein expression. Incubate at 18°C overnight at 200 RPM. IPTG is used to induce protein expression when the gene of interest is controlled by the lac operon and a T7 promoter. Depending on your construct, this might be different. Temperature and total time of protein expression needs to be optimised for individual proteins. Inducer concentration (IPTG here) also needs to be optimised. - Collect the induced cells by centrifuging at 4500 RPM for 25 min. Discard supernatant and either store pellet at -20°C or proceed directly to the next step. - Cells pellets left too long at -20°C can lead to protein degradation or aggregation of expressed protein. Keep the cell pellet no longer than 1 week at -20°C. **Protein Purification (without SUMO tagged protein)** Cell lysis carried out using the Retsch Mill machine. Cylinders and metal beads should be pre-cooled in liquid nitrogen before use. 1.Resuspend cell pellet in 5mL of binding buffer. (Optional: add 25 μL of 1 M MgCl2, 50 μL 200 mM CaCl2, 100 μg of DNase and 25 μg RNase) Add cell suspension into pre-cooled Retsch mill cylinders. Close the cylinder and snap freeze in liquid nitrogen for ~ 1 min Add the metal bead on to the top of the frozen cell suspension and shake the cylinder on mill for 2 min at 30/s frequency. After shaking, snap freeze the cylinder again, and repeat shaking two more times, freezing in between each shaking. - Collect the powderised cell lysate in a 50 mL falcon tube and resuspend in 25 mL binding buffer. Vortex until powder has dissolved and the lysate is homogenous. - Centrifuge at 15,000 g for 30 mins at 4°C. Collect the supernatant into a new tube and keep on ice. Discard the pellet. - Prepare 2 mL of Ni-NTA resin by adding it to the bottom of a gravity flow column. Wash column twice with 15 mL sterilised H2O, letting waste flow out of the bottom of the column. Wash once with 15 mL binding buffer. Apply a stopper to the end of the column and add the supernatant from step 3. Parafilm the top of the column and incubate at 4°C with slow stirring for 30 min - 1 hour. - Remove parafilm and stopper and let the unbound protein flow through the column. - Collect a sample of unbound fraction for SDS-Page analysis. - Wash column once with 20 mL binding buffer, then twice with 25 mL washing buffer. 1. Ensure that the majority of liquid has ran through the column before adding in more for the next wash. 2. Do not however let the column and resin run completely dry. 3. After every wash, small samples of flow through should be taken for SDS-Page analysis. - Elute proteins from resin by adding 10 mL of elution buffer. Repeat 2-3 times, collecting all the flow through. Change buffer to storage buffer via desalting column or concentrator tube. Adjust concentration to desired amount. Concentrate the samples to appropriate volume (500 μL) in a spin column. Check concentration of the protein on Nanodrop. Freeze at -80°C. Take a final sample from concentrated protein solution for SDS-Page analysis. **Purification of the resin for reuse:** - incubate 30 min with 10-15 mL of elution buffer, then elute- incubate 30 min with some 0.5 M NaOH- wash with ster. water- store in 5-10 mL of storage buffer- “The HisLink™ Resin and packed column may be used to purify protein more than once (generally two or three times), but we strongly recommend that the resin only be used for the same protein.” **Recipe sufficient for about 2 gels** Clean everything with EtOH Separation Gel: 4.1 mL H2O 3.3 mL Acrylamid (30%) 2.5 mL 1.5 M Tris-HCl, pH 8.8 100 µL 10% SDS 10 µL TEMED 32 µL 10% APS About 5 mL of separation gel solution per gel Overlay with Isopropanol and let polymerize (about 30 min) Discard Isopropanol, add stacking gel Stacking gel: 3 mL H2O 650 µL Arcylamind (30%) 1.25 mL 0.5 M Tris-HCl, pH 6.8 10 µL TEMED 20 µL 10% APS Don’t forget the camp Let the gel polymerize for at least another 30 min Mix samples with Loading Dye, denature samples for 5 min at 90°C, fill samples in wells, don’t forget the standard ladder Put voltage to 100 V until the front reaches the separation gel (about 15 min) Increase voltage to 120 V Let electrophoresis run until the Dye reaches the bottom of the gel (about 1.5 h) Stain gel in Coomassie brilliant blue solution for 2-3 h Destain gel in solution of 20% Methanol and 10% acetic acid overnight (or alternatively put gel in dest. water and boil for about 15 min in microwave)

### Standard Assembly

**1. Restriction digest insert and vector with appropriate enzymes:**

500 ng part/ plasmid and add

H2O until the total volume was 42,5 mL. Add

5 mL NEBuffer 2

0.5 mL BSA added to each tube.

* 1 mL first restriction enzyme added to each tube.
* 1 mL second restriction enzyme added to each tube.

The total volume was now 50 mL. The restriction digests were incubated at 37°C for

30 minutes, followed by an incubation of the digests at 80°C for 20 min to deactivate

restriction enzymes.

**2. Run vector and insert on a gel:**

The digests were put on a 1 % agarose gel and the electrophoresis was

performed with a voltage of 45 minutes.

**3. Ligate insert and vector:**

* 11 mL H2O
* 2 mL from each of the digests were added to the tube
* 2 mL T4 DNA Ligase Reaction Buffer
* 1 mL T4 DNA Ligase (total volume 20 ml).

The reaction mix was incubated at room temperature for 30 minutes,

followed by an incubation at 80°C for 20 minutes.

**4. Transform ligation product into competent cells see (transformation protocol.)**

- Screen colonies for correct plasmid by colony PCR (see colony PCR protocol)
- Miniprep DNA from chosen colony see miniprep protocol
- Make glycerol and sequence DNA

### Transformation

1. Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet

up and down several times, let sit for a few minutes. Resuspension will be red

from cresol red dye.

2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice,

and pre-chill 1.5ml tubes (one tube for each transformation, including your

control) in a floating fo a tube rack.

3. Thaw competent cells on ice: This may take 10-15 min for a 260μl stock.

Dispose of unused

competent cells. Do not refreeze unused thawed cells, as it will drastically

reduce transformation efficiency.

4. Pipette 50μl of competent cells into 1.5ml tube: 50μl in a 1.5ml tube per

transformation.Tubes should be labeled, pre-chilled, and in a floating tube

rack for support. Keep all tube on ice. Don’t forget a 1.5ml tube for your

control.

5. Pipette 1μl of resuspended DNA into 1.5ml tube: Pipette from well into

appropriately labeled tube. Gently pipette up and down a few times. Keep all

tubes on ice.

6. Pipette 1μl of control DNA into 2 mL tube: Pipette 1μl of 10pg/μl control into

your control transformation. Gently pipette up and down a few times. Keep all

tubes on ice.

7. Close 1.5ml tubes, incubate on ice for 30 min: Tubes may be gently

agitated/flicked to mix solution, but return to ice immediately.

8. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam

tube rack. Place in water bath to ensure the bottoms of the tubes are

submerged. Timing is critical.

9. Incubate on ice for 5 min: Return transformation tubes to ice bucket.

10. Pipette 950μl SOC media to each transformation: SOC should be stored at

4°C.

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# Results

### *E. coli* lab

To obtain the desired proteins, we successfully performed PCRs and constructed the plasmids via Golden Gate Assemblies (GGAs). As we wanted to investigate whether the efficiency of the Cytochrome P450 enzymes (CYP) increases when fused to certain Cytochrome P450 reductases (CPR) due to an improved electron transport between both enzymes, our aim was to construct plasmids for expressing the fusion proteins. (compare Fusionproteins Parts BBa\_K3846200-220) Plasmids for the expression of both proteins separately served as controls (e.g. BBa\_K3846368). For this purpose, we tried to fuse the CYP-enzymes and reductases in different combinations. The success of the assemblies was verified by sequencing.
Successfully assembled constructs:

* OleT-BM3R-Fusion* OleT-BM3R-Control* OleT-CPR- Control* CYP1A1-BM3R-Control* CYP1A1-Ferredoxin-Fusion* CYP71AV1-BM3R-Fusion* CYP71AV1-BM3R-Control* CYP71AV1-CPR-Fusion* CYP71AV1-CPR-Control

Unsuccessfully assembled constructs:

* OleT-CPR-Fusion* OleT-Ferredoxin-Fusion* CYP1A1-BM3R-Fusion* CYP1A1-CPR-Fusion* CYP1A1-CPR-Control* CYP71AV1-BM3R-Fusion* CYP71AV1-BM3R-Control* CYP71AV1-CPR-Fusion* CYP71AV1-CPR-Control* CYP71AV1-ATR2-Fusion

As shown above, numerous constructs could not be assembled successfully, although the according primers were redesigned several times.

Furthermore, the list of assembled constructs displays that there is only one combination of CYP-enzyme and reductase that was successfully assembled as fusion protein as well as a control containing both enzymes separately. Constructs that were only available as fusion protein or control could not be used to obtain significant data to confirm or refute our hypothesis. Reason for this is the requirement of a direct comparison of experimental results obtained with the fusion-protein on the one hand and results obtained with both enzymes separately on the other hand would be mandatory.

Nevertheless, our plan was to test the activity of CYP1A1 in combination with BM3R (unfused) to see if our experimental setup is working, as we hoped that we are going to have the corresponding fusion-protein soon afterwards. We wanted to use CYP1A1 for catalyzing the reaction of 7-ethoxycoumarin to 7-hydroxycoumarin. To monitor the reaction, we first investigated the emission of both substances at different wavelengths after excitation at 325 nm to make sure that we can detect the product in fluorescence measurements.

![](https://static.igem.org/mediawiki/2021/d/d5/T--Hamburg--res-1.png )

Figure 1: Fluorescence measurement of 7-ethoxycoumarin and 7-hydroxycoumarin after excitation at 𝜆 = 325 nm.
The fluorescence measurement revealed that both substances do have different emission maxima which do not not overlap (Figure 1). While 7-ethoxycoumarin reaches the emission maximum at a wavelength of 385 nm, the maximum for 7-hydroxycoumarin is 455 nm. Next, we used this information to investigate the enzyme activity. We used *E. coli* BL21 expressing CYP1A1 and BM3R separately to first measure the emission at 452 nm (emission of 7-hydroxycoumarin) of the cells without adding any substrate and then measured the emission again after adding 7-ethoxycoumarin. The difference in emission then should give the information whether 7-ethoxycoumarin is converted into 7-hydroxycoumarin or not. Additionally, we carried out the same experiment with wild type *E. coli* BL21 as a negative control.

![](https://static.igem.org/mediawiki/2021/e/eb/T--Hamburg--res-2.png )

Figure 2: Fluorescence measurement at 452 nm of *E. coli* BL21 with (left) and without (right) CYP1A1 and BM3R before (red) and after (blue) adding 7-ethoxycoumarin. The experiment was carried out with four different concentrations of the substrate resulting in different intensities of the emission.
The performed experiment showed that both - the cells carrying the plasmid for expressing CYP1A1 and BM3R and the wild type cells - converted 7-ethoxycoumarin into 7-hydroxycoumarin (Figure 2). The intensity in both approaches was nearly identical, indicating that the additional expression of CYP1A1 and BM3R did not influence the turnover of 7-ethoxycoumarin into 7-hydroxycoumarin. We were not able to carry out the experiment with the corresponding fusion-protein as the construct could not be assembled successfully.

Moreover, we wanted to use the combination of the CYP-enzyme OleT and the reductase BM3R for *in vitro* experiments as the construct for the fusion-protein (GGA 1.2) and for the control (GGA 3.1) could be assembled successfully. All proteins were designed to contain a 6x-His-tag at the N-terminus to allow purification via an affinity chromatography using a Ni-NTA resin. To confirm a successful protein expression and purification, samples of the different purification steps during affinity chromatography were collected and analysed via SDS-PAGE. As no clear bands were visible in those gels (which was indicated to be possible by an expert of CYP-expression (Prof. Dr. Vlada B. Urlacher) beforehand), we tried to detect the proteins of interest in a dot blot experiment using Anti-His-Antibodies.

![](https://static.igem.org/mediawiki/2021/0/00/T--Hamburg--res-3.png )

Figure 3: Dot blot of purified GGA 1.2 (OleT-BM3R fusion-protein) and GGA 3.1 (OleT and BM3R separately). Pink dots indicate the presence of a His-tag.
The dot blot confirmed that the proteins were expressed successfully and were present in the sample obtained after affinity chromatography (Figure 3). Next, we wanted to test the activity of the purified proteins by monitoring the conversion of myristic acid to 1-tridecen via mass spectrometry (EI). The obtained spectrum did not indicate the presence of the desired product. The most likely reason for this observation is that we were not able to properly dissolve the used carboxylic acid in anything but highly concentrated acetic acid and enzymes tend to dislike solid bulks of substrates and solutions of low pH.

Furthermore, we wanted to use CYP71AV1 to produce artemisinin. For this purpose, we used additional plasmids (Keasling group) containing the Mevalonate pathway. Those were necessary to produce precursors (amorphadiene) needed for artemisinin production catalysed by CYP71AV1. To check whether the needed precursors were produced, we transformed *E. coli* BL21 with the Keasling-plasmids and induced the expression of the proteins involved in the Mevalonate pathway. Afterwards, we used dodecan to extract possible contained amorphadiene and investigated the sample via mass spectrometry. After realising that no substance of interest was present in the spectrum, we soon discovered the reason why. We lacked a plasmid responsible for the expression of amorphadiene synthase which is needed to convert FPP to amorphadiene. Due to time limitations, we were not able to correct this mistake and therefore did not obtain any results using this construct.

FPP supplied by the mevalonate pathway can furthermore be used by α-humulene synthase to produce α-humulene. Long story short, this didn’t work out either.

### Cyano lab

The cultivation of cyanobacteria poses many obstacles, mainly due to slow growth. It is very important to keep wild type cultures of cyanobacteria axenic, as many bacterial and fungal contaminants have the potential to outgrow cyanobacteria. Therefore, the cultures were examined for contamination before three-parental-mating. This was done by plating out cultures on LB-agar plates and visually checking the cultures with a transmitted-light microscope. As pictured below, no contamination was observed, all colonies appeared uniformly coccal and greenish (Figure 4).

![](https://static.igem.org/mediawiki/2021/a/a0/T--Hamburg--wtSynechocystis.png )

Figure 4: Wild type *Synechocystis* *PCC 6803*, transmission-light microscopy.
Conjugation was executed using three-parental-mating with the helper strain *E. coli* *HB101* with plasmid pRK2012.2 and *E. coli* DH5a with PAM5411, coding for GFP as a three-parental-mating method control. Even though an unexpected tissue-localized-like fluorescence was observed in the unicellular organism *Synechocystis PCC 6803,* a fluorescence in colony-appearing structures was also seen, indicating a successful test-conjugation (Figure 5). After further investigation with confocal microscopy, it became obvious that no conjugation of the cyanobacteria occured, but one of the *E. coli* strains seemingly took up the construct. This was concluded as successful conjugates should show both red chlorophyll autofluorescence and GFP fluorescence, resulting in a yellow signal in the overlay picture. No such combined fluorescence was detected, rendering the conjugation protocol as not functional (Figure 6). Due to a very slow growth rate, no optimisation could be implemented.

![](https://static.igem.org/mediawiki/2021/8/8c/T--Hamburg--ThreeParentalMatingSamples.png )

Figure 5: Three-parental-mating samples, red fluorescence binoculum.

![](https://static.igem.org/mediawiki/2021/6/64/T--Hamburg--ThreeParentalMatingSamplesConfocal.png )

Figure 6: Three-parental-mating samples, confocal microscopy.

### Software tool LEA

To predict linker sequences we successfully built the software tool LEA (Linker Extraction from Alignments). We were able to show that the results are reasonable linker sequences that could be used for fusion protein design. Nevertheless, we were not able to carry out experiments proving an enhanced protein activity by modeled linker sequences due to time limitation. More detailed information can be found on the software tool page.

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