Biotechnology & Recombinant DNA Flashcards
what are the two things needed to make a recombinant DNA
two different sources of DNA to form a new DNA molecule - plasmid and gene of interest
what are the steps of genetic manipulation & cloning
- amplify a gene from organism A 2. Insert gene into plasmid 3. insert plasmid into organism B
how do you amplify a gene for genetic manipulation
use PCR - key here is taq polymerase because it is thermostable DNA polymerase that can handle high heat without denaturing and then extend the gene after heat
how do you insert a gene into a plasmid
through restriction enzymes (EcoRi endonuclease) - cuts after the G and before the A on both strands of the plasmid and the foreign DNA (strands are palindromes of each other so same on both strands) - the cohesive ends (sticky ends) of both the plasmid and the gene anneal - ligase seals the nicks
how have scientists prevented the plasmid from annealing back with itself during gene manipulation
created a genetically modified plasmid for cloning - done through a multiple cloning site (region with lots of restriction enzymes in a row) - additionally always encode for a positive selection marker (usually antibiotic resistance to select for only the bacteria that got the gene)
how does lacZ help with cloning plasmids
the functioning lacZ gene will produce a bacteria that has a blue color to it - non functioning will be white - so the colonies with the plasmid inserted into the cloning region can be selected for
what do transcriptional and translational fusions do for science
allow the discovery of when things are activated in a cell
how does transcriptional fusion work
an insertion of a gene + its ribosome binding site are inserted into the target gene - when the target gene is expressed so is the inserted gene (usually insert something that can glow so easily traceable)
how does translational fusion work
the inserted gene does not have its own ribosome binding site so the target protein fused with the inserted gene
what is the difference between transcriptional and translational fusion
translational fusion allows the inserted gene to be regulated by the same things as the target protein - so if wherever the target protein is localized then the inserted gene will show that
what is fur / when is the operon expressed
the ferric uptake regulator that regualtes genes based on iron concentration / when iron is available the operon is repressed - when starved the operon will be expressed
what is the innate response / what is the adaptive response
fast and nonspecific / a slower but more specific - remembers specific viral infection and defends
what is the innate response of bacteria / how do bacteria protect themselves from their innate response
restriction modification systems / they methylate their own DNA as the eco ri is nonspecific and will chop up any invading sequence
what is the adaptive response in bacteria / how do bacteria have an adaptive response
the CRISPR cas-9 system / they have to modify their genome and pass that down to their offspring
what is the PAM site / how does bacteria use it to protect themselves from their adaptive response
the PAM sight is in target DNA - what the CAS1 and CASII scan for in incoming phage DNA / DNA of bacteria does not contain PAM sights but does contain the spacer and a repeat to match future phage DNA
