Lecture 18 Flashcards
What is a restriction enzyme? What are the normal functions of restriction enzymes in bacteria?
Why do they not cut the host chromosome?
restriction enzymes that cut ds DNA are enzymes that cleave a vector such that recombinant DNA can be inserted
Their normal functions in bacteria are to act as an innate immune system and protect it from invading nucleic acids
bacterial chromosome is methylated which allows restriction enzymes to recognize the host chromosome and not cleave it
Define vector in the context of recombinant DNA technology.
a vector is a circular carrier molecule of DNA and has its own origin of replication such that it can replicate by itself.
Why is it true that the use of a single restriction enzyme on DNA from two sources will yield multiple possible ligation products? Why is this not going to happen in directional cloning?
When DNA is cleaved by restriction enzymes it has complementary sticky ends so without directional cloning the DNA would reanneal / self-ligate into the original vector
this does not happen in directional cloning as you use 2 restriction enzymes such that the sticky ends are not complementary so the vector can only anneal in one direction
Why is directional cloning a much better option for cloning into expression vectors as compared to
using a single restriction enzyme?
directional cloning is better for expression vectors(vectors to express gene from a different species) that have a promoter on one need and a termination sequence on one end. If the insertion of the gene of interest can be either way, there will be some transcripts that are wrong so using two restriction enzymes makes it such that cloning occurs in one direction.
You are cloning a human gene responsible for the sequestration of heavy metals in the cytoplasm. You
use a pUC-based plasmid. Following the construction of your recombinant vector, your transformation
only yielded blue colonies. What does this indicate?
The lac z gene when plated on an antibiotic plate will produce blue colonies if functional.
The multiple cloning site is within the Lac Z gene, so once the gene of interest is inserted into the plasmid the Lac Z gene will not be functional, and instead of blue colonies being produced white colonies will be produced.
Since the transformation only yielded blue colonies that means the ccbacteria did not receive recombinant plasmids and the gene of interest was not properly inserted into the plasmids.
What is the key difference between a plasmid and a BAC?
The key difference between a plasmid and a Bacterial Artifical Chromosome is that BACS can accommodate much larger fragment of DNA for eukaryotic genes that are larger and cannot be inserted into plasmids.
Suppose you sequence all clones in a gDNA and a cDNA library derived from the same organism.
In the gDNA library, you find sequences corresponding to gene A are three times as abundant as
sequences corresponding to gene B. However, in the cDNA library there are three times as many
sequences corresponding to gene B compared to gene A. Propose a plausible explanation for this
pattern
gDNA is reflective of the copy number of a sequence meaning there quantitatively there are3x as more sequences for gene A than gene B.
However cDNA reflects the expression level ( due to it being DNA created from reverse transcribing RNAs) so while there may be an abundance of gen seqs compared to gen B seqs, gene B seqs are still being expressed at a higher level. Additionally genes A and B respond to different environmental conditions and therefore must have different levels of expression.
You are trying to express a human gene in bacteria. You find that no matter how strongly you
induce expression of the human gene, there is very little protein produced and some of your data
seem to indicate that the protein is not folding properly in its bacterial host. This is in stark contrast to
the amount of mRNA for this gene being produced, which is very high. Propose a plausible reason for
this pattern and devise a solution
this is due to the codon bias that bacteria have where the bacteria has specific trnas that are not being recognized by the human codons. This results in misfolding and low expression
to fix this you can use codon optimization:
synthesize a synonymous protein sequence from the bacteria that is synonymous to the codon that is transcribed for the human gene of interest.
Suppose the genetic code were not universal. What complication would this introduce in terms of
recombinant DNA technology? Would it make heterologous gene expression impossible? Explain
if the genetic code were not universal you would not have synonymous codons across species and therefore you could not use expression vectors to express genes of one species in another. so heterologous gene expression would be impossible.
What problem does recombinant DNA technology solve regarding diabetes and growth disorders
in humans?
By inserting the gene isulin like gene into bacteria via recombinant technology there will be a lot of insulin produced that could be used for those with diabetes. This was far safer than extracting insulin from the pancreas of pigs and cattle which could potentially lead to an allergic reaction.
What is the role of opines in the life cycle of Agrobacterium transformans?
Agrobacterium transformants is a bacteria that cause tumors on plants. These bacteria care the TI plasmid which contains t-dna
T-dna causes the plant cells to divide uncontrollably and to produce opines which and be used as nutrients by the bacteria to grow on the plant but not be used as food by the plant itself . RESULTS: BACTERIA TURN PLANT INTO FOOD FACTORY
Compare and contrast the F plasmid with the Ti plasmid.
The TI plasmid contains genes that encode homologous machinery to that used to transfer DNA in F plasmids.
Explain how you would use a two-plasmid approach to introduce a Bt toxin gene into corn. What
genes would be on each plasmid? How would you isolate transformed cells?
a disarmed Ti plasmid with removed tumor gene and opines
the second plasmid is a transformation plasmid containining gene of interest selectable marker and border sequences. ‘
you would isolate transformed cells by indeitify which one s have the slectable marker
What is the consequence of illegitimate recombination relative to homologous recombination?
illegitimate recombination results in the location of introduced genes not being easily controlled. As a result if a gene is integrated in an unwanted location it could lead to a frame shift or point mutation
Why are transgenic salmon kept as triploids? Why is there variance in the size phenotype among
transgenic salmon?
triploid offspring are sterile so this prevents the very large transgenic salmon from repopulating in the wild.
eggs are injected with a growth hormone gene where the regulatory sequence has altered to be constitutive as opposed to being expressed sometimes.
There is variance in the size phenotype w. transgenic salmon is due to illegitimate recombination that results in the gene being inserted in heterochromatic region in one salmon in euchromatic regions in others which means the gene will be expressed more when inserted in euchromatic regions.
pressure-treating the egg prevents a 2nd meiotic division resulting in diploid eggs that once fertilized by sperm will yield triploid offspring
