Team:UCL/Project/Experiments

Here we explain some of rthe experiments and procedures we undertook. The details of our procedures are presented in our protocols page.

Creating Competent Bacteria

E. coli are not naturally transformable, which means they lack the ability to take up plasmids (competency). Competency is induced by divalent cations such as calcium. These alter the permeability of the membranes enveloping the bacterium to plasmids. Normally macromolecules on the outer surface of bacteria are negatively charged which means the negative charges of incoming DNA would be repelled. The addition of calcium chloride facilitates the movement of DNA into the cell.

Transformation

To insert foreign DNA into our competent cells we used the heat shock treatment. Our competent bacteria are stored in -80C. To transform, DNA with a selectable marker and competent bacteria are mixed together and kept on ice for thirty minutes to allow interactions between calcium ions and the negative charges on the bacterial envelope. The mixture is exposed to a brief period of 38C (heat shock). The rapid shift in temperature alters the fluidity of the membrane therefore allowing DNA to enter the cell. Afterwards, the bacteria containing the foreign DNA are streaked on selective plates. Bacteria containing the foreign DNA with the selectable marker, such as ampicillin resistance, would be the only bacteria growing on the selective plates.

Minipreparation

This step involves purifying plasmid DNA from bacteria. This allows us to prepare a stock of DNA ready for digest, ligation or further transformation. This is done by first lysing the cells then applying centrifugation on the sample in spin filters. Eventually after the process DNA would be purified as it elutes through the filter along with elution buffer.

Analytical Digest

Gel electrophoresis exploits the fact that the negatively charged DNA fragments would move in a electric field. Furthermore, in the gel matrix, smaller fragments of DNA would move with more ease. This allows separation of shorter fragments of DNA from longer fragments. Our DNA sample is first digested into fragments with restriction enzymes. Finally the digested DNA sample is added to the gel. After exposure to an electric field for an hour, separated fragments can be observed under UV.

Nanodrop

The nanodrop, or spectrophotometer, quantitatively measures the purity of DNA in a sample. It uses the fact that nucleic acids absorb UV light in a specific pattern. The Beer-Lambert Law is used to determine the concentration of DNA as a function of absorbance.

Preparative Digest

This experiment is used to build up a stock of a specific fragment of DNA. Afterwards, the digested sample is run on gel electrophoresis. Compared to the analytical digest, this is done in larger quantities of DNA sample and enzymes. To isolate the wanted DNA fragment we extracted the band with the correct length on the gel.

Gel Extraction

This procedure is used to extract the DNA fragment, with the expected band, on the gel. With the use of UV light, DNA bound to ethidium bromide illuminates and can be carefully extracted from the agarose gel. The removed fragment of gel would contain the desired DNA fragment. Furthermore, DNA would have to be purified from the gel fragment using a gel purification procedure. This DNA could be utilized for ligation, transformation and so on.

Polymerase Chain Reaction

The polymerase chain reaction uses enzymes and primers to generate a large quantity of copies of a DNA sequence. This allows us to amplify a gene or sample to a more workable quantity. In addition, with the correct use of primers, we would be able to modify a gene so that illegal sites could be mutagenised and addition of the iGEM standardized prefixes and suffixes.

Ligation

If an insert needs to be attached to a backbone (vector) containing a selectable marker, a ligation is used. Insert and backbone will need to have compatible sticky ends prepared from the preparative digest. Ligase, Ligase buffer, correct conditions are used in the process to catalyse this reaction.

Maxipreparation

Similar to a minipreparation, the maximpreparation is used to purify DNA. The maxipreparation, however, produces more pure and higher concentrations of DNA.

Zeocin Kill Curve

Before doing a transfection experiment, it is important to determine the concentration of selection reagent required for efficient selection. For this, we growed HeLa cells in various concentrations of Zeocin - 0 nM, 50 nM, 100 nM, 250 nM, 500 nM and 1000 nM. We conducted the kill curve in a 6-well plate. A T25 flask of HeLa is split in 10 ml medium. Each well is filled with 1 ml of cells. Incubation is carried out at 37C overnight to allow cells to attach before adding inhibitor (Zeocin). Medium is removed and replaced with 2 ml of DMEM + Glu + FBS + Zeocin. Confluency of cells is observed in each well every day for 5 days. Viability can be assessed using a Vicell machine, which stains dead cells blue.

HeLa Growth Curve

We conducted a growth curve for HeLa cells in a 6-well plate. A T25 flask of HeLa is split in 10 ml medium. Each well is filled with 1 ml of cells. Incubation is carried out at 37C overnight. Medium is removed and replaced with 2 ml of DMEM + Glu + FBS. Confluency of cells is observed in each well every day for 5 days.

Transfection

Hela cell culture (0.1x106 cells/ml) was seeded in four 6-well plates 24 hours prior to transfection to attain 70% confluency. When cells reached 70% confluency they were transfected. Control flasks are transfected with only plasmid backbone pSB1C3.

5ug of BBa_K1018001 plasmid DNA (12.5ul of a 401 ng/ul stock) was added to 5ml of DMEM supplemented with 2mM L-glutamine only in a 15ml falcon tube. The solution was vortexed briefly and incubated at room temperature for 5 minutes. 20ml of a 1mg/ml branched 25KDa polyethylenimine (or SuperFect) was added to the solution before vortexing briefly and a further 5-minute incubation at room temperature.

The media in the cell culture flasks was aspirated and replaced with the transfection solution and cells were returned to incubation conditions. Control flasks were treated with 5ml DMEM supplemented with 2mM L-glutamine and 20ml of branched PEI (or SuperFect) as above.

After 24 hours the media was replaced with DMEM supplemented with 10% FCS, 1% PenStrep antibiotic, 2mM L-Glutamine and 150mg/ml Zeocin.

Medium is removed and replaced with 2 ml of DMEM + Glu + FCS + Zeocin. Confluency of cells is observed in each well every day for 5 days.

Amyloid Degradation Assay

The 42-amino acid peptide (Aβ1-42), the predominant peptide length found in senile plaques, has a remarkable propensity to aggregate at high concentrations to form a β-pleated sheet structure. While plaques and amyloid fibrils have been viewed by some as resistant to proteolytic degradation, it is possible that certain proteases, such as MMP-9 may contribute to endogenous mechanisms leading to plaque clearance. Our assay, inspired by Yan et al., demonstrates our BioBrick's capability to do this.

Congo Red Assay

The birefringence version of this assay was originally developed to study amyloid in vivo but has since been expanded into frequent in vitro use. It depends on the birefringence of amyloid; an optical property of a material that has a refractive index dependent on the polarisation and propagation of light (i.e. orientation differences within the molecule in question), meaning that there is a double refraction of light. Many materials can be birefringent, including phosphate buffer, and this assay is subjective, so a known fibrillar material needs to be used as a control. The spectroscopic version of this assay is less subjective and less prone to misinterpretation, and involves recording absorbance spectrums.