Exam 3 pt 2 Flashcards
insulin is made with bacteria process
- The two different polypeptides (A chain and B chain) are inserted into different plasmids and then put into bacteria (transformed).
- The cells are cultured. Purified B-galactosidase-insulin fusion proteins.
- Treat with CNBr. CNBr cleaves the peptide bond after methionine.
- purify A and B chains.
- Refolding and disulfide bond formation
the cycle threshold is reached when
the accumulation of fluorsecense is much greater than the background fluorescence detected by a thermocycler
Why do many researchers include GADPH in their Northern Blot experiment?
Because GAPDH is expressed at the same level in all cell types, researchers include it in their Northern blot experiments as a control. It confirms that the same amount of RNA is present for each cell type and that the Northern blot was successful.
Why are The A and B chains are linked to 𝛽-galactosidase in production of insulin
This is done so that the fusion protein is stabalized. If the chains were not linked to 𝛽-galactosidase, then they would be degraded, and 𝛽-galactosidase increases stability.
why are The purified proteins are treated with cyanogen bromide in insulin making
This is done to cleave off the beta-galactosidase portion of the protein. This cleaves the peptide bond connecting the fusion proteins to separate them into separate proteins. This allows the protein to form into insulin and fold properly.
biological control
the use of living organisms or their products to alleviate plant diseases or damage from environmental conditions.
* Ex: Bt is natural occurring and kills caterpillars and insects but is nonharmful to humans and plants. Put on crops
bioremidation
the use of microorganisms to reduce enviromental pollutants
Ex: microorganisms to treat and degrade sewage
biotransformation
enzymes produced by the microorganism transform the structure of the toxic pollutant
how is a gene knockin made in mice
- gene of interest is cloned and flacked with peices of DNA that are homologous to a noncritical site in the mouse genome
- cloned DNA introduced into a mouse oocyte
- gene of interst inserts into the noncritcal site by homologous recombination
gene reduncy might mean
that a gene knockout has no effect
procedure of making a protein be screted in milk
- insert gene in plasmid next to milk specific promoter
- DNA injected into sheep oocyte
- female transgenic offspring secrete protein in milk
- purify protein from milk
cloning of animal how
- donor sheep’s mammary cell is extracted and grown in flask
- another sheeps unfertilized egg is extracted and nucleus removed
- mammary cell and egg with no nucleus are fused with electrical pulses
- egg starts turning into an embryo with donor DNA and egg proteins
- embryo put in surrogate
- identical sheep born
dolly age problems
seemed to be as old as donor bc shorteing of telomeres matched the donor
stem cells characteristics
- capacity to divide
- differentiate into anything
totipotent cells
can give rise to all cell types
pluripotentent
can turn into most cell types
multipotent
several cell types can turn into
unipotent
can only turn into one cell type
hematopoietic stem cells are
multipotent
most ES cells from
IVF
most EG from
aborted fetuses
induced pluripotent stem cells
made pluripotent, but how to make them differentiate?
DNA microarray is
plastic slide with many sequences of DNA
microarray can
identify genes that are transcribed
DNA fragments on a microarray can be
- amplified by PCR and then spotten onto the microarray
- synthesized directly on the microarray itself
how is microarray used to study gene expression
- islate mRNA
- make cDNA that is fluorescently labeled
- hybridize cDNA to microarray
- view on a lser scanner
*
microarrays make it possible
to study the expression of the whole genome under different enviromental conditions
goal of ChIP
to determine where target protein binds in genome
ChIP steps
- add formaldehyde to crosslink proteins to DNA
- lyse the cells
- sonicate DNA into small peices
- add antibodies that recognize the protein of interest
- the antibodies are bound to heacy beads
- centrifuge the sample
- pellet contains protein of interest, linked DNA
- uncrosslink the pellet
two options:
* if have an idea of DNA: conduct PCR of DNA
* if PCR amplifies then the protein of interest was bound to the DNA region that is flanked by the primers
OR
- attach linkers
- conduct PCR with linkers and flurescence
- do microarray
RNA seq used to
study the simultaneous transcription of many genes
transcriptosomes
the set of all RNA molecules that are transcribed in one cell or a population of cells
RNA Seq technique
- isolate RNA
- break RNA into small samples
- add linkers to the RNA
- synthesize cDNA with reverse transcriptase PCR
- sequence cDNA
- align the cDNA sequences along the genomic sequence
RNA seq advantages over microarrays
- more accurate at quanitfying the amoung of each RNA
- superior at detecting RNA in low abundance
- shows exact boundries between exons and introns
- identifies 5’ and 3’ end of RNA transcripts
gene knockout collections allow
researchers to study gene function at the genomic level
recognition strategies
- sequence recognition
- pattern recognition
recognition strategies
- sequence recognition
- pattern recognition
search by signal
tries to locate an organization of known sequence elements that are normally found within a gene
search by content
tries to identify sequences that differ significantly from a random idstribution due to codon bias
open reading frame searching
looking for a region that does not contain any stop codons
homolous genes
are derived from the same ancestral gene