Development of a reverse split-ubiquitin system to characterize protein-protein interactions

2. Material and Methods:

2.1 Yeast strains:

The following yeast strains were used for the split ubiquitin system.

Tab.1: Yeast strains for the transformation. Shown are the yeast strains and its genotype with which the yeast transformation was performed.

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2.2 Yeast transformation

YAPD medium was used as a growth medium for the strains AH109 Clontech and BY4741b. This was shaken overnight at 30°C at 200 rpm until the stationary phase. Subsequently, the medium was diluted 1: 5 in fresh YAPD medium and stirred for a further 3 hours (hr) at 130 rpm. Meanwhile, 2 mg/μl salmon sperm (Invitrogen, #15632-011) DNA was boiled in the heating block at 99°C for 15 minutes and put immediately on ice. A DNA Mix was prepared. For this, 800 ng of each plasmid were needed for the transformation and used in a final volume of 34µl. To enhance the transformation efficiency 240µl PEG 3350 (50%) and 36µl lithium acetate (1M) were added.

The Yeast was centrifuged shortly at 8000 rpm. 50 µl of the boiled salmon sperm DNA was resuspended with the DNA Mix. The Yeast was centrifuged at 8000 rpm again and the cell pellet was overlayed with the DNA Mix containing the boiled salmon sperm DNA and vortexted shortly. The Mix was incubated at 42 °C for 1:30 hr at 500 rpm.

The cells were centrifuged at 800 rpm for 30 seconds. The pellet was resuspended very carefully with 800 µl H2O. 400 μl of the cells were applied to SC plates containing no leucine and tryptophan. (See App. SC Medium). Glass beads were used to cover the cells on the plates. The plates were incubated for at least 2 days at 30 °C.

2.3 Polymerase Chain reaction:

For the Polymerase Chain Reaction (PCR) KAPA High Fidelity (KK2601, Biosystems) and Phusion high-fidelity DNA polymerase (F530S, ThermoFischer Scientific), Polymerase, were used as described by the manufacturer. The primers used are shown in Appendix, Tab. 6.

Tab.2: PCR approach with KAPA High Fidelity DNA Polymerase.

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Tab.3: PCR cycle parameter for KAPA High Fidelity DNA Polymerase

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Tab.4: PCR approach with Phusion high-fidelity DNA polymerase.

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Tab 5: PCR cycle parameter for Phusion high-fidelity DNA polymerase.

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2.4 Ligation into the pGEMT vector

The pGEMT vector system (Promega, Mannheim) is very suitable for cloning PCR amplificates into a vector. To check whether the PCR product was incorporated into the vector, the blue white selection was used.

2.5 Cloning into target vectors:

For the preparative digestion always a 20 µl batch was used. This included 2-3 µg DNA template, 2 µl 10x buffer and 0.5 µl enzymes (10 µg / µl) and H2O.

After addition, the digest was incubated for 2 hours at 37°C and applied to a 1% agarosegel electrophoresis. The bands were cut out with a scalpel under UV light and extracted according to instructions from the manufacturer (GenElute ™ Gel Extraction Kit, Sigma Aldrich).

2.6 Bacterial Transformation

Table5: Used bacterial strain with its genotype.

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For the transformation of the ligation mixtures by heat shock competent DH5a cells were used, which were previously thawed on ice. The ligation mixture was added to the cells and incubated for 30 minutes on ice. They were exposed to a heat shock of 42°C for 45-50 seconds and placed on ice for 2 minutes. Subsequently, liquid LB medium without antibiotic was added and shaken at 37°C for 1,5 hr if necessary. The plating was carried out on LB selection plates, which were incubated overnight at 37°C.

2.7 DNA Mini-Preparation

Overnight cultures are scheduled the day before the mini preparation. Each of them contains a bacterial colony in 5 ml LB medium with the appropriate antibiotic and are then stirred at 37°C overnight in the incubator at 200 rpm. 4 ml of this overnight culture were harvested the next day by centrifugation at 6000 rpm for 3 minutes in an Eppendorf Centrifuge. The mini preparations were made according to instructions from the manufacturer PureLink™ Quick Plasmid Miniprep Kit (Invitrogen). To get a better amount of DNA, sequencing verified plasmids were purified according to the instructions of GeneJET Plasmid Miniprep Kit (#K0692, ThermoFisher Scientific™). Concentrations were measured with the NanoVue™ Plus Spectrophotometer (GE Healthcare). All constructs were screen in control digestion (see Fig. 8,10,12 and 14) and sequenced before further usage.

2.8 Control digestion

To screen the minipreparations, a test digestion was performed. For this, 500 ng DNA from each Minipreparation were cut with selected enzymes and analyzed in agarose gel electrophoresis.

2.9 Sequencing

To check the cloned vectors for correctness, they were sequenced according to the "Sanger" method at Eurofins Genomics in Ebersberg. The samples were prepared according to the manufacturer's instructions. The nucleotides used for this are listed in the Appendix in Table 2.

3. Results

3.1 Creating vectors with TEF1 promoter

3.1.1 GLO-1 and C35B1.2a

To develop a special Yeast Two Hybrid System, the “bait” and “prey” genes were first cloned into the appropriate vectors containing a TEF1 promoter. glo-1 (QL) was used as prey protein and was cloned into a pNub frame 2 vector which containes the N-terminal Ubiquitin. The glo-1 (QL) form is a dominant active form. Over time, several effector proteins interacted with the protein. One of them is C35b1.2a, which was cloned into a pDB Trp vector and act as bait protein (R.Häfner, personal communication). This vector also contains the C-terminal Ubiquitin (Fig.7).

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Fig.8: pDB Trp vector with a TEF1 promotor and C35B1.2a as the gene of interest. It also contains the C-terminal Ubiquitin, followed by a glycine-glycine-arginine sequence and the reporter gene His3.

Subsequently, a test digestion was practiced checking whether the gene was completely cloned into the vector. In the test digestion for bait, restriction enzyme MluI was used. Here two bands were expected at 7631 bp and 730 bp for positive clones. For prey, three bands were expected at 3887 bp, 3484 bp and at 1274 bp. (Fig. 8A). For pNub frame 2 – glo-1(QL), the gel showed the expected bands. It also showed one other band at under 2000 bp. It was then sent away for sequencing. In the test digestion of the pDBTrp-TEF1-C35B1.2a, restriction enzyme MluI was used. Here two bands were expected at 7631 bp and 730 bp. Again, the gel showed the expected bands (Fig. 8B).

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Fig.9: Test digestion of pNub frame 2- glo-1(QL) and pDBTrp TEF1 – C35B1.2a applied on a gel electrophoresis. (A) Test digestion with EcoRV. Three Bands were expected at positions 3887 bp, 3484 bp and at 1274 bp. The gel showed the three expected bands for correct insertion. It also showed one other band at under 2000 bp. (B) Test digestion with MluI. For correct insertion two bands were expected at positions 7631 bp and 730 bp. The gel showed the two expected bands.

3.1.2 C35B1.2a (Aa95-301)

In previous analysis the interaction domain of C35B1.2a to GLO-1(QL) could be narrowed down to Aminoacids 95-301 (R. Häfner, personal communication). We also wanted to analyse this Interaction in the Reverse Split-Ubiquitin System. For this, it was cloned into the pDB Trp vector with TEF1 promotor (Fig. 9). For test digestion restriction enzyme Bsp119I was used. For positive clones, two bands at 6412 bp and 892 bp are expected. The gel showed two bands (Fig.10).

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Fig. 9 Created pDB Trp vector with a TEF1 promotor and C35B1.2a (Aa 95-301) as the gene of interest. It also contains the C-terminal Ubiquitin, followed by a glycine-glycine-arginine sequence and the reporter gene His3.

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Fig. 10: Test digestion of pDB Trp - TEF1 – C35B1.2a (Aa 95-301) applied on a gel electrophoresis. For the digest the enzyme Bsp119I was used. Two Bands were expected at positions 6412 bp and 892 bp. The gel showed the two expected bands for correct insertion.

3.2 Creating vectors with SSA1 and SSB1 promoter

As it might be that the TEF1 promoter is leading to a strong expression (Th. Blume, 2016), we decided to express C35B1.2a also with weaker promoters, Stress-Seventy subfamily A1 (SSA1) and Stress-Seventy subfamily B1 (SSB1) (Fig.11).

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Fig. 11: pDB Trp vector with C35B1.2a as the gene of interest. It contains the C-terminal Ubiquitin, followed by a glycine-glycine-arginine sequence and the reporter gene His3 with a.) SSA1 promoter b.) SSB1 promotor

The promoters SSA1 and SSB1 were cloned into the pDB Trp - TEF1 - C35B1.2a Cub His3* Vector. For the SSA1 promoter, the enzyme CaiI was used for the test digestion and two bands were expected at 7626 bp and at 946 bp. The gel showed the expected two bands (Fig. 12A). For the SSB1 promoter, the enzyme Bsp119I was used in the test digestion. Upon successful cloning, 3 bands were obtained at 5758 bp, 1675 bp and 997 bp. The gel showed the three bands (Fig. 12B).

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