Experiments

Geneious Prime: A bioinformatics software platform used for sequence analysis, this enables scientist to test restriction reactions, gibson assembly, design primers and other.

Alphafold: A protein structure database that allows to troubleshoot recombinant proteins in silico. This allows to test if modified proteins fold an maintain their function.

Benchling: A bioinformatics software platform that can be used for sequence alignment in addition to other sequence analysis tools.

PyMol A molecular visualization system for molecular modeling, in our case of our CRISPR system

By use of primers designed to exclude a specific region in a plasmid, a plasmid can in large part be amplified and made into a linear copy. This also produces appropriate overhangs that were designed to function with our construct G blocks. The resulting PCR product of this linearization contains the desired linear sequence, with compatible overhangs with out G-block constructs. After PCR to confirm that the linearization reaction had worked, a restriction digestion reaction was set up with both the PCR product and non-linearized plasmid. A restriction endonuclease is chosen that has a restriction site in the plasmid but which is lost after linearization. After the digestion is complete this reaction is subjected to gel electrophoresis and analyzed.

Then the PCR products were cleaned and the concentration was measured using Nanodrop to move on to Gibson assembly.

This method is based on 3 enzymatic activities:

1. Exonuclease activity – creating ssDNA overrhangs for dsDNA that can then anneal to a ssDNA complement (in this case our g blocks).
2. DNA polymerase activity – for filling in the gaps after annealing of our ssDNA and the g blocks.
3. DNA ligase activity – covalently sealing nicks in our assembly.

It’s an efficient way to join and clone DNA molecules larger than 300 kb into cloning vectors (in our case pASK-IBA2). These constructs were sequence verified and then cloned.

BamHI – restriction endonuclease, was used to digest the circular pASK-IBA2 vector as a control. The linearized pASK-IBA vector has lost the BamHI restriction site.

After fragments and linearized vector (pASK-IBA2) were subjected to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour were transformed into TOP10 cells. TOP10 cells are useful for clonning due to their high transformation efficiency [1].

Heatshock transformation is a method used to introduce foreign DNA into bacteria. In our case we introduced pASK-IBA2 vector with our constructs: HLC and CLS. BL21α cells were transformed in order to start protein expression experiments. BL21α are used for expression for non-toxic heterologous genes and is an efficient expression strain [2].

Small-scale expression was conducted in order to optimize protein expression conditions.
We decided to test our construct expression in 16°C, 30°C, 37°C and room temperature (22-24°C) and inducing the expresion after 1 h, 3h and over night.

Large-scale expression was conducted after optimization (30°C degrees overnight induction) to move on to purification.

Recombinant HLC protein has 6 Histidine residues chosen to purify the construct under Ni-NTA affinity purification of a bind-wash-elute procedure.

N-terminal His-tagged protein (HLC) was purified on a nickel-nitrilotriacetic acid (Ni-NTA) HisTrap™ FF 1 ml column (GE Healthcare) with a sepharose matrix pre-charged with nickel ions. The column was equilibrated in 5 column volumes (CV) buffer A (see buffer recipes – his-tag purification buffer a). Before the filtered supernatant oF HLC wasloaded onto the column using a 50 ml superloop (GE Healthcare). The column was then washed with 10 CV buffer a (see buffer recipes – his-tag purification buffer a) before HLC was eluted during a 0-100% gradient of buffer b (see buffer recipes – his-tag purification buffer b) over 15 CV.

Fractions from the chromatography are analysed on an sds gel.

A Strep-tag is a synthetic peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and was fused to our protein for efficient purification using an affinity column.

Bis-Tris Native gel [3]

The native gel system we used has a near neutral pH (~7.5) to perform native (non-denaturing) electrophoresis. This environment results in better band resolution due to the stability of the protein and the gel matrix.

The NativePAGE ™ Gel system is based on the Blue Native Polyacrylamide Gel Electrophoresis (BN PAGE) technique developed by Schägger and von Jagow (Schägger & von Jagow, 1991) that uses Coomassie G-250 as a charge-shift molecule [1]. After binding to the proteins the charge shift molecule confers a negative charge but does not denature the protein – leaving it in its native state. The Coomassie G-250 is in the cathode solution that is applied to the gel continuasly.

Is a method to separate proteins based on their size, all the proteins in a complex mixture are denatured and have a negative charge and they are separated by size while they move towards the positive probe.

Agarose electrophoresis

Gel electrophoresis method used to separate macromolecules such as DNA, RNA and protein in an agarose matrix. Proteins can be seperated by their isolelectric charge (not applied in our project), DNA and RNA can be seperated by length.

Gel-Red staining

An intercalating molecule that when bound to nucleic acid and exposed to UV light strongly fluoresceces. It is safer due to being less toxic than ethidium bromide.

Silver staining

A staining method that has a sensitivity in the nanogram range. The working principle is silver ions binding to the protein that are then reduced under appropriate conditions that results in a visible image from a fine coat of silver.

Analytical chromatography

Since we did not succeed in visualizing our CasMINI proteins binding the RNA with native gel or agarose gels we decided to run our samples through an analytical column and overlap the resulting chromatograms. In theory, gRNA only sample, CasMINI only sample and gRNA bound to CasMINI sample should have different sizes, which is reflected in chromatograms after running them through the column.