The project

Many molecular biology techniques take a significant amount of time to complete. Many of the enzyme-based reactions which underpin these techniques require incubation periods of and hour or more, while cloning and transformation often requires overnight incubation to allow the transformed cells time to recover and multiply. In the limited time we have available, we cannot hope to cover all molecular biology techniques, however you will undertake a mini-project which will expose you to some of the more important DNA-based techniques.

In this project, you will be provided with a culture of Escherichia coli (E. coli), a bacterium which is widely used in molecular biology. This culture has been transformed (genetically engineered) by the introduction of a small loop of DNA (a plasmid) which contains a gene of interest. Your task is to use a technique called a mini-plasmid preparation (mini-prep) to recover this plasmid from the culture and demonstrate its presence using electrophoresis.

Getting started

Before you begin, make sure that you are familiar with the relevant theory behind the techniques we will be performing. This manual contains several appendices which will provide you with this information. Make sure you read this information before proceeding.

  • Appendix A : DNA

Other molecular biology techniques are provided at the SPARQ-ed website here.

Theoretical basis of the project

Using plasmids for cloning

One of the more common techniques available to scientists working in molecular biology is cloning. In this technique, sequences of DNA containing genes of interest are inserted into vectors which are then used to introduce these genes into cells or organisms to study the effects of the expression of the genes.

Vectors are based on bacterial plasmids – short circular pieces of DNA separate to the main bacterial chromosome which may be transferred between bacteria. Scientists source plasmid vectors from biological supply companies, which create them by ligating together pre-existing genes and sequences of DNA built from scratch using sequencing technology. A map of an example of a commercial vector (the pGEM-T Easy system) is presented in the figure below.

Map of the pGEM-T plasmid vector

Note that this map shows a number of regions contained within the vector. The numbers refer to how many base pairs along the sequence (out of a total of 3015) a particular region is found.

In our experiment, the pGEM-T Easy vector has had a short fragment of the gene for the protein polo-like kinase 1 (PLK1cloned in at the insertion site (located at around “3 o’clock” if you imagine the picture of the vector to be a clock face). This portion of the gene codes for a region of the protein called the polobox domain, which assists in the localization of the protein at various point during the cell cycle. Information about how this gene fragment was cloned into the pGEM-T vector can be found here.

Prior to today’s project, this cloned vector was used to transform E. coli cells. The cells were used to inoculate an agar plate containing ampicillin and the plates incubated overnight. Because of the presence of the ampicillin resistance gene in the pGEM-T vector, the only cells to grow into colonies were those that had been transformed by the plasmid. These colonies were then used to inoculate a culture broth, which has been provided to you. 

The alkaline lysis mini-plasmid preparation

Using transformed bacteria is a very efficient means to generate DNA needed for research. Bacteria have minimal requirements for nutrition and so the production of large quantities of DNA can be done quickly and at a minimal cost. Once a culture of transformed cells is established, this culture can be used to seed new cultures, so transformation need only be performed once. However once we have our culture, we need a way of recovering the DNA in a relatively pure form.

DNA (including plasmid DNA) is not generally secreted by cells. In order to recover it, we need to disrupt the cells and then purify the DNA we need from the other cellular contents. This is the purpose of the alkaline lysis mini-plasmid preparation (or mini-prep).

The first stage of the mini-prep involves bursting the cells using an alkaline solution. This releases their contents into the surrounding liquid. An acidic solution is then added, which neutralizes the alkaline solution and denatures the proteins, causing them to become insoluble. They can be removed from the cell lysate through centrifugation (see figure below).

 

Mini-prpe procedure - lysis

The second stage of the mini-prep involves passing the cell lysate through a column. These columns bind onto plasmid DNA, and allow chromosomal DNA and other cell products to pass through. After washing the column several times, we can make the column release the plasmid DNA by passing through an elution solution (see figure below).

Mini-prep - extraction of plasmid

Restriction digests

By the end of the mini-prep procedure, you should have approximately one drop of a colourless liquid. To demonstrate that you have recovered the plasmid DNA you will need to run the sample on an electrophoresis gel. However, before your sample is ready to run, you must first prepare it using a restriction digest.

Plasmid DNA is circular. For DNA to be demonstrated on a gel, it needs to be linearised. This is done by using enzymes to cut the DNA (think of cutting a rubber band once to obtain a straight strip of rubber). Restriction endonucleases are enzymes which cut the DNA strand at very specific locations, normally given by sequences of half a dozen or so base pairs called restriction sites. If we know the sequence of a length of DNA, we can select enzymes which cut the DNA once (ie. the restriction site sequence occurs once in the entire DNA sequence) or even twice. If a plasmid is cut twice, you should end up with DNA fragments of two different sizes. Molecular biologists often use this double cutting to “drop out” an insert they have cloned into a vector.

The pGEM-T Easy vector has been created with a number of restriction sites on either side of the insertion point. Some of these restriction sites are only found on one side of the insertion point, and so can be used to linearise the vector to make it ready for electrophoresis. Others are found on both sides, and so may be used to drop out the insert to check its size. In this exercise, we will be using the enzyme EcoRI which has restriction sites just upstream (before) and downstream (after) the insertion site. This will allow us to drop out the polobox insert, resulting in DNA of two different sizes : around 3015 base pairs long for the vector, and 800 base pairs long for the insert. The two linearised fragments are now ready to be demonstrated using electrophoresis.

Further information on restriction digests can be found here

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Agarose gel electrophoresis

If we wanted to sort sand from gravel from larger rocks, we would use a series of sieves of different sizes. Each sized sieve lets smaller particles pass through but retains the larger fragments. Electrophoresis can be thought of as a sieve for large molecules like DNA or protein.

In agarose gel electrophoresis, a DNA sample is loaded towards one end of a block of a jelly-like substance called agarose. When an electrical current is passed through the gel, the DNA molecules are pushed through the gel away from the negative electrode (DNA has an overall negative charge and like charges repel). Smaller fragments of DNA can move more easily through the gel than larger fragments, so in a given period of time, DNA of different sizes accumulates in regions of the gel. If we include a dye which binds to the DNA, , these regions are visible as bands – the further towards the positive electrode a band is located, the smaller the fragments of DNA are found in that band.

To get an idea of the size of a band seen on a gel, we always run a sample consisting of a mixture of DNA fragments of known sizes alongside our test samples. This is called a marker, or a “ladder”, as the multiple bands of DNA seen on the gel resembles the rungs on a ladder. By matching the position of a band in our test sample to those representing DNA of known size in the ladder, we can estimate the size of DNA fragments in our test. The part of the PLK1 gene which codes for the polobox domain is 800 base pairs (bp) long. Therefore, if our digest has been a success, we should see a band corresponding to our DNA markers which is 800 base pairs long representing the polo-box insert, and another representing the pGEM-T vector at 3000 base pairs long.

More detailed information on electrophoresis can be found here