Here you can find the protocols we engineered and followed to conduct our lab experiments. That way, they are easily accessible for anyone who wishes to replicate our research! Note: Protocols may have to be adapted if using different bacteria strains.
Transformation is the process of adding desired plasmids into a chemically competent cell. The bacterial cell is incubated with the plasmids on ice, transferred to intense heat for a short amount of time (to make the cell membrane semi permeable) then put on ice again to help the cells recover with the plasmids inside them.
When colonies have correctly grown on plate, a liquid culture needs to be grown up of a single colony to create a monoclonal culture for plasmid extraction and growth of further cultures for protein expression or similar uses.
To confirm that the correct plasmids have been added to the cells, the plasmids are extracted again to run on an agarose gel with a ladder to see if the plasmids are the correct size. This is also a way to multiply and extract more plasmids if running out.
Once plasmids have been extracted, the concentration of DNA extracted needs to be quantified, so the Qubit Fluorometer is used, alongside standards to acquire the concentration.
Restriction digest is the process of cutting up the plasmids before and after (at the promoter and terminator) the desired assembled genes, to create fragments that can be identified in an agarose gel. The plasmids are cut by enzymes, such as EcoRI and PstI.
An agarose gel is a way to separate DNA fragments by size, like the desired genes and plasmid backbone. The fragments formed are negatively charged, so when they are run with a current, the smaller fragments move faster through the gel towards the positive terminal. The result is bands that should be in line with the expected size of the fragment on the ladder.
Once the plasmids have been added into the bacteria, they need to be activated to express the proteins. This is done through using autoinduction media, which uses glucose to grow up the concentration of cells, then lactose to induce high expression of the gene, and produce lots of desired protein.
To harvest the protein, the cells must be broken down both chemically and mechanically to extract all the protein but also not destroy it. The cell lysis digests all the parts of the cell that are not required like the cell membrane and DNA and should leave the protein intact.
Nickle columns are used in protein purification due to the ability of histidine-tagged proteins to bind to nickel reversibly. However, between uses for different proteins, nicked columns need to be 'recharged' by stripping and reattaching nickel ions to the silica beads.
Proteins with histidine tags that bind to nickel bind reversibly- a pH controlled buffer containing imidazol can be used to displace the his-tags. When a high concentration of imidazole is added, the nickel ion binds to the imidazole instead of the histidines. When performing a protein purification using a nickel column, some non-desirable proteins may loosely bind to the nickel beads due to having naturally occurring histidine. Therefore, two buffers (containing a lower and higher concentration of imidazol respectively) are prepared- one to displace loosely bound background proteins, and one to elute the desired protein from the column.
The amino acid histidine has an affinity for nickel, meaning that proteins containing a histidine (his) tag will reversibly bind to nickel. Nickle columns contain nickel bound to silica beads, which cell lysate or other protein-containing solutions can be ran through to separate the protein from the solution. The column can be washed (removing loosely bound non-target proteins) then eluted, producing (hopefully) pure fractions of the desired protein for further analytical work.
A protein qubit is an analytical technique used to determine the concentration of protein in a sample by using target-selective dyes that fluoresce when bound to protein. Using the standards, a standard curve is established by which the samples are compared.
Western Blots are used to visualize, distinguish, and quantify proteins in a sample, so effectively to make sure the protein of interest is present. Proteins are separated by size via gel electrophoresis, before being transferred to a membrane and incubated with antibodies specific to the protein of interest. A secondary antibody is added alongside staining to visualise the protein- the thicker the band, the more present.
Restriction digests use naturally occurring enzymes that cleave DNA at specific sequences. This produces DNA fragments with compatible 'sticky' ends to be inserted into the plasmid (vector), in this case into our genes of interest (cas-12a and cas-13). Before this occurs, the fragments of DNA are separated by weight into bands via gel electrophoresis in an agarose gel, ready for excision and ligation.
After completing an agarose gel with your restriction digest DNA fragments, you can physically remove the bands from the gel and break it down in order to extract the DNA for ligation. Ligation is the reforming of the parts of DNA to insert the gene into 1 complete plasmid.