synthetic biology Flashcards
How is new DNA naturally introduced to bacteria ?
bacteriophages inject DNA into bacterial cells
Explain what competent cells are
cells that have been chemically altered so that they are more likely to take up foreign DNA
define transformation
the process by which genetic material is transferred between bacterial cells
define plasmids
circular, double stranded DNA molecules
how many base pairs do plasmids usually contain
few thousand
by what lab techniques are plasmids manipulated before undergoing transformation in to bacterial cells
restriction enzyme digestion and ligation
what are the key features of an expression plasmid ( aka expression vector )
1) promoter - upstream of gene of interest, can drive transcription
2) immediately down stream of promoter there are sequences that allow bacterial ribosomes to translate resulting mRNA for gene of interest
3) translation starts at initiation codon (ATG)
4) site to clone gene ( MCS) a series of unique restriction enzyme sites
5) transcriptional terminator downstream of MCS
what is a restriction enzyme ?
protein produced by bacteria that cleaves DNA at specific sites along molecule
what is the frequency of which a restriction enzyme will cut DNA a function of
the length of of the sequence it recognizes
what formula is used to calculate the cutting frequency of restriction enzymes
(1/4)^n n= the number of contiguous base pairs in the recognition site
what type of ends to restriction enzymes produce
blunt end - no overhang
staggered end - creates sticky ends that are much easier to manipulate during cloning because of their base pairing capacity
what are the primer pair constraints when designing a primer
- It must span the target
- needs to be unique
- similar Tm
- point in the right direction
- not be complimentary
what are the primer constraints
- no hairpins
- ideally have a GC clamp ( 3’ ending in G or C )
What technique is used to isolate plasmids from bacteria
alkaline lysis
what are the key steps to follow when carrying out alkaline lysis
1) Harvesting bacteria from culture
2) suspension of bacterial pellet in resuspension buffer
3) lysis of bacteria in alkaline buffer
4) neutralization of lysate
5) clearing of lysate
6) recovering plasmid from cleared lysate
7) washing of plasmid DNA
8) Storage of plasmid DNA
how is the plasmid DNA separated from from the chromosomal DNA and the proteins ?
adding the alkaline buffer causes a denaturation of the molecules. when the solution is neutralized and brought back down to pH7 the plasmids renature first with a higher probability than the chromosomal DNA
this step is kept short so that only the plasmids are able to build double helices and become soluble again. the tube is centrifuged to separate the insoluble proteins and Chromosomal DNA from plasmids.
why do we wash and centrifuge the miniprep spin column after the neutralization step
to remove excess salt
how are restriction digests analyzed
gel electrophoresis
what three conformations can plasmid DNA exist in?
supercoiled, open-circular or linear
in what conformation do plasmids exist when inside a cell
supercoiled
what happens if there is a break within one of the strands of DNA in a plasmid
DNA will remain circular but the the break will allow for movement around the phosphodiester backbone and the supercoil will be released
what what plasmid conformation will travel further down a gel
supercoiled
what is useful to have when ligating two pieces of DNA together
to have a similar number of ends
what are the key steps of transformation ?
- competent cell preparation
- Transformation by mixing DNA with competent cells
- recovering
-plating
