Experiments


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.


Plasmid to Protein Experiment Pipeline

Transformation Protocol

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.

Liquid Culture Preparation Protocol

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.

Plasmid Extraction Protocol

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.

DNA Quantification using Qubit Fluorometer

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 Protocol

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.

Running an Agarose Gel Protocol

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.

Autoinduction Media for overexpression of Protein Protocol

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.

IPTG Induction for overexpression of Protein Protocol

Once the plasmids have been added into the bacteria, they need to be activated to express the proteins. This is done through addition of the chemical IPTG to induce high expression of the gene, and produce lots of desired protein.

Lysis of Expressed Bacteria Protocol

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.


Protein Purification, Quantification and Identification Protocols

Column Recharging Protocol

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.

Buffer Preparation Protocol

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.

Protein Purification Protocol

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.

Protein Qubit Protocol

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 Blot Protocol

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.


Fluorescent Probe Protocols

Environmental RNase Contamination Protocol

Testing Different Enzyme Concentrations for RNase Alert

Testing Different Probe Concentrations for RNase Alert

Testing Different Enzyme Concentrations for DNase Alert

Testing Different Probe Concentrations for DNase Alert

Fluorescein Concentration Curves Protocol


Transcription Protocols

PCR Amplification of Targets and T7 in vitro Transcription Protocols


Other Protocols

MBP Restriction Digestion Protocol

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 (Cas12a and Cas13a). 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.

Gel Excision, DNA Extraction and Ligation Protocol

After completing an agarose gel using restriction digest DNA fragments, bands can be physically removed from the gel to break it down and allow DNA to be extracted for ligation. Ligation is the process of re-forming of parts of DNA by inserting a gene into 1 complete plasmid.

Reducing Blood Opacity Protocols

Reducing Synthetic Blood Opacity Protocol

Cas13a Final Proof of Concept Protocol

This experiment was conducted as a final proof of concept for our Cas13a-based diagnostic test. The results of this experiment can be viewed here. This protocol could be adapted for any complimentary crRNA and target pair.

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