Color Your Classroom: Engaging Students with Bacteria and Bio-Art
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Origin of Replication: a DNA sequence from which bacteria can initiate the copying of the plasmid. Multiple Cloning Site: a short DNA sequence that contains many unique restriction enzyme sites and allows scientists to control the introduction of specific genes into the plasmid. Promoter: a DNA sequence that is typically located just before (“upstream” of) the coding se- quence of a gene. The promoter recruits RNA polymerase to the beginning of the gene sequence, where it can begin transcription.
Selectable Marker
Promoter
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Multiple cloning site
Plasmid Map
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Origin of Replication
Figure 2: Plasmid Features
4. Selectable marker: a gene that codes for resistance to a specific antibiotic (usually ampicil - lin, kanamycin or tetracycline). When using selective media, only cells containing the marker should grow into colonies, which allows researchers to easily identify cells that have been successfully transformed. TRANSFORMATION EFFICIENCY
final vol at recovery (mL) vol plated (mL)
Number of transformants µg of DNA
Number of transformants per µg
X
=
In practice, transformation is highly inefficient—only one in every 10,000 cells successfully incorporates the plasmid DNA. However, because many cells are used in a transformation experiment (about 1 x 10 9 cells), only a small number of cells must be transformed to achieve a positive outcome. If bacteria are transformed with a plasmid containing a selectable marker and plated on both selective and nonselective agar medium, we will observe very different
100 transformants 0.01 µg Specific example: X
100,000 (1 x 10 5 ) transformants per µg
1 mL 0.1 mL
=
Figure 3: Bacterial Transformation Efficiency Calculation
results. Nonselective agar plates will allow both transformed and untransformed bacteria to grow, forming a bacterial “lawn”. In contrast, on the selective agar plate, only transformed cells expressing the marker will grow, resulting in recovery of isolated colonies. Because each colony originates from a single transformed cell, we can calculate the transformation efficiency, or the number of cells transformed per microgram (µg) of plasmid DNA (outlined in Figure 3). For example, if 10 nanograms (0.01 µg) of plasmid were used to transform one milliliter (mL) of cells, and plating 0.1 mL of this mixture (100 microliters, or 100 µL) gives rise to 100 colonies, then there must have been 1,000 bacteria in the one mL mixture. Dividing 1,000 transformants by 0.01 µg DNA means that the transformation efficiency would be 1 X 10 5 cells transformed per µg plasmid DNA. Transformation efficiency generally ranges from 1 x 10 5 to 1 x 10 8 cells transformed per µg plasmid.
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