lead to multiple drug-resistance phenotypes in the recipients.
Ampicillin belongs to a group of antibiotics known as the penicillins. Members of this group act as competitive inhibitors of a step in the synthesis of the bacterial cell-wall component.β-lactamase is a protein, which degrades ampicillin, and thus gives rise to amipicillin resistance in bacteria, which contain the β-lactamase gene. This enzyme is released into the extracellular medium by the bacteria, and thus degrades ampicillin molecules in the vicinity of the cell.(1)
The ampicillin gene works by bacterial transformation
Transfer of genetic material between bacteria via DNA molecules.
When plated on a petri dish on an agar based medium:
Each colony is an asexual clone of a single plated individual.
Generation time is rapid. Millions of asexual offspring are produced. E.coli is prokarote. It therefore has no membrane bound nucleus as in an eukaryote. Instead it has a circular piece of continuos DNA (its chromosome and also its linkage group). It does not however have chromosome proteins or centromeres.
Antibiotic resistance:
Normal medium verses antibiotic medium. Bacteria can maintain DNA as plasmids: circles of DNA that usually contain a gene that allows the bacterium to grow in the presence of an antibiotic. When a plasmid is introduced into bacteria it is called transformation.
Bacteria are treated so they will take the plasmid up into their cells. These are called competent cells.(6)
Materials and methods:
The E. coli strain used in this study is MM294. The pAMP plasmid, which codes for ampicillin resistance, was used in trasnformations.
Two agar plates were provided:
- LB agar plate with untransformed cells (wild type)- the control. (-)
- LB/amp agar plate with transformed cells (E.coli)- experimental. (+)
Single colonies of genetically identical cells were isolated from the two plates using the loop and then were streaked onto two other plates containing an agar/ampicillin medium.
The bases of the plates were labelled accordingly (+amp, -amp).
The plates were then taken and placed upside down in an oven.
In the second part of the experiment practise pipefitting was done onto filter paper to produce spots. The diameters of the spots were compared and the percentage accuracy of the pipetting was calculated.
Ready prepared fragments of DNA of various sizes were provided ready to load onto a gel electrophoresis column. The column was run and the results collected. The sizes of the fragments were then finally estimated.
Results
The following results were obtained for the first part of the experiment:
The wild type did not grow, while the E.coli containing the gene for antibiotic resistance did. These results are what I would expect (the pictures of this are shown below).
The pipetting practise produced spots with diameters of different sizes according to the different volumes pipetted out. (See below).
For 2.5microlitre the average spot diameter is 9mm. For 2microlitres the average spot diameter is 0.8-0.9mm
For 1micrlitre the average spot diameter is 8mm. (As the volume decreases the spot diameter also decreases).
% Accuracy of pipetting:
A- over by 2.42 microlitres out of ten.
B- over by 3.9 microlitres out of ten.
C- over by 3.3 microlitres out of ten.
Therefore:
A- 24.2 % accurate
B- 39 % accurate
C- 33% accurate
This is not a very good accuracy and can be improved upon with more practise.
Below I have shown a picture of the results of running the DNA fragments through the gel electrophoresis column:
Estimation of size of fragments using the 1kb ladder:
Table:
Graph:
Discussion:
WHY THE BASE AND NOT THE LID?
The lid can be swapped and then a case of mistaken identity becomes apparent. By labelling the base it is clear which sample is which.
WHY ARE THE PLATES PLACED UPSIDE DOWN?
If there is any condensation on the lid it does not fall onto the agar and lyse the bacteria.
WHAT IS THE REASON FOR HAVING AMPICILlIN IN THE PLATES?*
The reason for having ampicillin in the plates is to ensure the growth of bacteria containing the ampicillin resistance gene and replication of the corresponding plasmid so that colonies become visible.
(allows selection of the transformed bacteria).
WHY PERFORM THIS PARTICULAR TYPE OF STREAKING PATTERN?
The most common way of separating bacterial cells on the agar surface to obtain isolated colonies is the streak plate method. It provides a simple and rapid method of diluting the sample by mechanical means. As the loop is streaked across the agar surface, more and more bacteria are rubbed off until individual separated organisms are deposited on the agar. After incubation, the area at the beginning of the streak pattern will show confluent growth, while the area near the end of the pattern should show discrete colonies. The difference in growth appears due to gradients of oxygen and nutrients and toxic products. At the edge nutrients are plentiful, at the centre there is a thicker layer of bacteria and the required oxygen and nutrients diffuse less readily into the centre. Their toxic products can not be quickly eliminated and hence cells on the periphery grow at maximum rates.(3)
WHAT ARE THE ADVANTAGES OF BLUESCRIPT OVER Pbr322 AS A PLASMID CLONING VECTOR?
The bluescript has multiple restriction sites. The Pbr322 vector does not.
WHAT IS THE BIOLOGICAL FUNCTION OF A RESTRICTION ENDONUCLEASE?
Restriction endonucleases provide an anti-viral protection for bacteria by cleaving the DNA of invading bacteriophages.
Different enzymes cleave at different positions within the recognition sequence, and may generate blunt fragments, or fragments with 5' or 3' overhanging ends.(2)
WHAT DOES THE GEL LOADING BUFFER DO?
The gel loading buffer has a buffering capacity at a fairly alkaline pH. This ensures the DNA is negatively charged so that it migrates smoothly from the well into the agarose. It also ensures all fragments of equal size migrate together over the same distance.
WHAT DOES THE TBE DO?
Salt ions in TBE allows the transfer of electrical charge between the gel electrodes. The ions reduce the resistance of the water and allows easier conduction of electricity. This ensures smooth DNA band migration through the gel. It prevents water from overheating.
WHAT ELSE DOES THE BLUE-BROMOPHENOL BLUE DYE DO?
Bromophenol dye is the most popular tracking dye used in polyacrylamide gel electrophoresis in neutral or alkaline buffer systems. It allows us to see the movemeof nucleic acid samples due to the coloured dye. It is used to show the distance the sample has migrated. The dye does not complex with DNA. (5)
WHAT DOES THE ETHIDIUM BROMIDE DO?
It allows visualisation of the nucleic acid in gel electrophoresis. Ethidium bromide is a DNA intercolator (complexes with DNA). The fluorophore is excited by light in the u.v. range and emitts electromagnetic radiation in the visible range. It is moderatley toxic and a potent mutagen and needs to be disposed of carefully.(7)
WHICH WAY DOES THE DNA MIGRATE?
The DNA migrates towards the positive electrode (the anode).
WHY DOES IT MIGRATE IN THIS DIRECTION?
The DNA is negatively charged at the oxygens within the phosphate groups within it, and is attracted towards the positive electrode. DNA has no positively charged molecules under neutral/alkaline pH conditions.
Conclusion:
The purpose of this technique is to introduce a foreign plasmid into bacteria and to use those bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.
References:
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- Microbiology-fifth Ed. Prescott, L. M., Harley, J. P., Klein, D. A. published by Hill, M. Page. 297
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- http://www.nmsu.edu
