Question 1. Although we will not be doing a gel electrophoresis, data from a gel digest of a Bacillus anthrax plasmid is provided so you can do a DNA map. The Bacillus anthrax plasmid is 4000bp (4Kb) long. Note the origin position as well as the reference molecular weight markers on the gel. Two restriction enzymes, A and B, were used to obtain two individual digests, A and B. They were combined to produce the third digest. The restriction enzyme fragment pattern for the digest of Bacillus anthrax plasmid Determining the Number of Fragments How many fragments were produced by enzyme A? How many fragments were produced by enzyme B? How many fragments were produced by the combined digest (A and B)? Fragment Size Fragment size is relative to molecular weight, and must be determined by comparing the fragment distance to the molecular weight markers. The fragment size has been provided on the gel pattern for this exercise. To make a map you must determine the relative positions of the restriction sites on the plasmid. It helps to know that the double digest is an extension of the two single digests. The bands you can see in the "A" digest are essentially then subjected to the "B" enzyme digest. Smaller band numbers seen in the double digest can be combined to add up to the "size" of the bands in the A digeStarting the Map Determine which two bands in the double digest make up the small A fragment. Mark the bands on the fragment line below, and indicate the size of each. These two bands represent where the B restriction site is on the fragment. Small A Determine which two bands in the double digest make up the large A fragment. Mark the bands on the fragment line below, and indicate the size of each. These two bands represent where the B restriction site is on the fragment. Large A Determine which two bands in the double digest make up the small B fragment. Mark the bands on the fragment line below, and indicate the size of each. These two bands represent where the A restriction site is on the fragment. Small B Determine which two bands in the double digest make up the large B fragment. Mark the bands on the fragment line below, and indicate the size of each. These two bands represent where the A restriction site is on the fragment. Large B Superimposing fragments to map the plasmid. To determine the relative position of the restriction sites on the plasmid, you need to align each of the fragments from the single digests to create one longer fragment, and superimpose one on the other.
Bacterial Genomics
The study of the morphological, physiological, and evolutionary aspects of the bacterial genome is referred to as bacterial genomics. This subdisciplinary field aids in understanding how genes are assembled into genomes. Further, bacterial or microbial genomics has helped researchers in understanding the pathogenicity of bacteria and other microbes.
Transformation Experiment in Bacteria
In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.
Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
Question 1.
Although we will not be doing a gel electrophoresis, data from a gel digest of a Bacillus anthrax
plasmid is provided so you can do a DNA map. The Bacillus anthrax plasmid is 4000bp (4Kb) long.
Note the origin position as well as the reference molecular weight markers on the gel. Two
restriction enzymes, A and B, were used to obtain two individual digests, A and B. They were
combined to produce the third digest.
The restriction enzyme fragment pattern for the digest of Bacillus anthrax plasmid
Determining the Number of Fragments
How many fragments were produced by enzyme A? How
many fragments were produced by enzyme B?
How many fragments were produced by the combined digest (A and B)?
Fragment Size
Fragment size is relative to molecular weight, and must be determined by comparing the fragment
distance to the molecular weight markers. The fragment size has been provided on the gel pattern
for this exercise.
To make a map you must determine the relative positions of the restriction sites on the plasmid. It
helps to know that the double digest is an extension of the two single digests. The bands you can
see in the "A" digest are essentially then subjected to the "B" enzyme digest. Smaller band numbers
seen in the double digest can be combined to add up to the "size" of the bands in the A digeStarting the Map
Determine which two bands in the double digest make up the small A fragment. Mark the bands on
the fragment line below, and indicate the size of each. These two bands represent where the B
restriction site is on the fragment.
Small A
Determine which two bands in the double digest make up the large A fragment. Mark the bands on
the fragment line below, and indicate the size of each. These two bands represent where the B
restriction site is on the fragment.
Large A
Determine which two bands in the double digest make up the small B fragment. Mark the bands
on the fragment line below, and indicate the size of each. These two bands represent where the A
restriction site is on the fragment.
Small B
Determine which two bands in the double digest make up the large B fragment. Mark the bands on
the fragment line below, and indicate the size of each. These two bands represent where the A
restriction site is on the fragment.
Large B
Superimposing fragments to map the plasmid.
To determine the relative position of the restriction sites on the plasmid, you need to align each of
the fragments from the single digests to create one longer fragment, and superimpose one on the
other. The alignment of the two large fragments from the plasmid digest is shown below.
Combined A and B Large Fragments
Large A fragment
A A (3) B 1.25 | 1.75 B
Large B fragment
B B (2.5)
A 1.75 | 0.75 A
Large A and B superimposed A B A B
| |
1.25 | 1.75 | 0.75Do an alignment below for the combined A and B small fragments
Small A fragment
Small B fragment
Small A and B superimposed
Align your two superimposed fragments (the superimposed large and the superimposed
small fragments) and look for a repeat.
You can now make a linear map of the plasmid.
The data from your linear map can be redrawn to form the circular plasmid with iQuestion 2. Reconstruct the circular restriction map of all three restriction enzymes (A, B and C) from the
information given below.
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