What is the purpose of DNA transformation?
-changing the phenotype
What are plasmids?
-plasmids occur naturally in bacterial cells -circles of DNA -self replicating -extra to the bacterial DNA -often when bacteria survive the presence of antibiotics= plasmids allow them to survive
first obtained from bacteria then engineered to have three principal components - origin of replication, gene for some sort of antibiotic resistence inserted(here ampR codes for enzyme beta lactamase that brakes down ampicilin), multiple cloning site- recognition sequences for lot of restriction enzymes so you can open up the plasmids, these sequences can be all over the plasmid as well
How are plasmids named eg: pBR322
p-plasmid BR- names of researchers 322- probably the number of attempt
How do you open a plasmid and insert sequence you want?
-with a plasmid- use restriction enzyme, open it up
-put in DNA cut with the same enzyme - then they will both have the same unattached bases so they will hybridise
-used extensively eg humulin- engineered human insulin
How are plasmids used with growth deficiencies?
if child not growing enough- give the growth hormone= growth (usually when child grows less than 5cm a year between the ages 3-12)
-2 types of growth hormone- put the hromone coding sequenc einto the plasmid then back to the bacteria the gene expressed and produce of growth hormone
-somatonorm- supplement that some athletes use to bulk up
(Turner's syndrome women given this to grow)
How are Haemophiliacs A helped with plasmids?
-can make factor VIII
-Factor VIII gene into plasmid, into bacteria and produce Factor VII for the treatment of Haemophilia A
How can we make bacteria resistant to Ampicilin?
most wild bacteria- not ampicillin resistant
can add the gene (make the peptidoglycan walls permeable to the plasmid) some will take the plasmid
-then test on agar with ampicilin: the ones with the plasmid will grow the rest won't- recognise which using the ampicillin= transformed bacteria
-not all bacteria will take up the plasmid= only small colonies
the plasmid in prac. can take the sensitive bacteria and expose them to the plasmid, some will take it, we recognise it beacuse they will glow in UV light!
lac Z gene- if grown on particular agar= blue if human gene put in= white colonies
-way of determining if the plasmid is taken up
-the gene does something we want but this way we know if it's taken up or not and which ones will produce the wanted substance
Where do we see interference RNA and what is it?
-in XIST inactivation
interference RNA- when RNA interferes with gene expression
What is messenger RNA?
-carrying the DNA molecule message into the cytoplasm for use
-formed during transcription from a DNA template
What is ribosomal RNA?
-rRNA and proteins combine to make ribosomes
-structural RNA-ribosomes= important for gene expression
pic=almost looks double stranded because of how it bend back on itself
What is transfer RNA?
-Carries amino acids to the ribosome in translation
-anticodon matches with codon of mRNA
-specific amino acid for each anticodon attached to 3’ end
-shuttles the amino acid into position during translation
parts of this RNA double stranded
What is micro RNA?
smaller, only 22 nucleotides in length
there may be a gene sequence not coding for a polypeptide only for RNA- get the micro RNA- can be complementary to mRNA that was going to produce a polypeptide and can bind to it and stop this sequence from working- can be permanent but also temporary eg. environmental factors
What is the central dogma of genetics?
-In bacteria the chromosome in cytosol so transcription and translation happen there almost simultaneously, in sequence
-in eukaryote more complicated, added step- processing, RNA must be modified before it is called mRNA- this happens in the nucleus,first produce primary RNA transcript then modification to mature mRNA
Describe the statistics of the human genome:
A gene consists of the coding sequence and regulatory sequences a gene is a sequence of DNA from 75 bp - 2 300 000bp (2300kb)
-Only one strand of the DNA double helix in a given gene codes for the polypeptide = template strand (can be either one)
5'-3' direction of transcription
so the it only adds bases to the 3'end