Lecture 1 Flashcards
biotechnology
the use of living organisms for industrial purposes
Genetic Engineering (molecular biotechnology)
the use of experimental techniques to produce DNA molecules containing new genes or new combination of genes
Overview of cloning project?
clone the gene for an ENHANCED variant of the green fluorescent protein (egfp), originally from Aequorea victoria, from a cloning vector into a CLONING VECTOR into an E. coli EXPRESSION VECTOR so that the jellying protein will then be made by the E. Coli bacteria
Plasmid
a circular piece of DNA, independent of the host chromosome, capable of replication
Vector
A DNA molecule (often plasmid) capable of replication in a host organism that is specially modified for propagating DNA sequences
Recombinant DNA
DNA created in vitro by joining together pieces of DNA that are not normally contiguous
Gene cloning
insertion of a fragment of DNA, carrying a gene, into a plasmid vector and the subsequent propagation of the recombinant DNA molecule in a host organism
fusion protein
protein made by splicing two genes or gene sequences together, which when translated, produce a hybrid of chimeric protein
sub cloning
cloned DNA that is moved from one plasmid to another
Typical Steps in Traditional Gene Clothing
Insert: isolate DNA of interest to be clones
vector: choose a suitable cloning vector for amplification and manipulation of foreign DNA
CUT: restriction endonuclease digestion of vector and insert (foreign) DNA
Ligate: covalently join vector and insert together (paste)
Transformation: introduce recombinant molecule into prokaryotic or eukaryotic cell to serve as host for amplification of DNA (e. COli is the most common host)
Select: identify cells that received a plasmid
Screen: identify cells containing recombinant DNA (clones)
Amplify: depending on the vector, large amounts of recombination DNA, RNA, or protein can be made
E. Coli plasmid (engineered and in nature)
AA
what are two types of commonly used plasmid vectors?
cloning vectors- are primarily used only to propagate DNA. Have everything necessary to insert foreign DNA and to replicate itself, but does not usually transcribe the inserted DNA
Expression vectors-are primarily used to make recombinant protein. Have everything necessary to insert foreign DNA and to replicate itself. Also contains everything necessary for gene transcription and translation.
Recombinant Proteins can be made using cloning vector?
FALSE
TorF: DNA can be inserted in an expression vector.
True
TorF: DNA is typically inserted into an engineered plasmid at the multiple cloning site.
TRUE
What are characteristics of a cloning vector?
- High copy number
- Cloning Site (multiple cloning site)-site where DNA is usually inserted
- Origin of Replication
- Selectable Marker
- screenable marker
What is a selectable marker vs a screenable marker
selectable marker: (antibiotic resistance)- only bacteria that have been TRANSFORMED WITH THE PLASMID containing the selectable marker will be able to grow. Plasmid alone or plasmid + insert
Screenable marker: all bacteria containing plasmid grow (due to selectable marker) but this allows you to distinguish the transformed bacteria that contain the insert in the plasmid front eh ones that do not.
What is the purpose of selectable marker?
- one medium containing the antibiotic, allows growth only of bacteria containing the plasmid and kills bacteria that do not contain the plasmid. (during a transformation procedure, selects for bacteria that received the plasmid)
- Allows you to maintain SELECTIVE PRESSURE by including antibiotic growth medium every time you grow the bacteria. Without selective pressure, plasmids are rapidly lost.
WHY would you not want to lose the selectable marker?
because you would lose the ability to select and maintain selective pressure
Characteristics of expression vector
have all necessary components to transcribe and translate your gene of interest.
**they are designed to express genes and make good proteins
- origin of replication
- selectable marker
- often lower copy number (but not always) than cloning vector
- PROMOTER (usually inducible)
- Ribosome binding site
- ATG start codon
- Cloning sites that allow inserts to ligate in a predictable reading frame and orientation
- often (not always) have a fusion tag to aid in purification of recombinant DNA
Alkaline lysis
separated bacterial chromosomal DNA from plasmid DNA by taking advantage of the SUPERCOILED nature and size of plasmid
-both plasmid and chromosomal DNA are denatured at a high pH brought about by addition of NaOH
Denaturation
hydrogen bonding is disrupted and double stranded DNA is separated into single strands
What are three similar steps that most plasmid purifications procedures start with?
- resuspend bacteria in buffer containing RNase
- Lyse bacterial membranes with SDS and denature DNA with NaOH
- Neutralize solution to reanneal plasmid DNA and precipitate out chromosomal DNA
What are the two purification processes that we use in lab?
alkaline lysis
silica adsorption
Anion Exchange chromatography
- is a type of ion-exchange chromatography
- negatively charged DNA will bind resin. Positively charged molecules flow through
- Elute DNA from column with high salt buffer
Silica adsorption
Disposable mini columns has a silica membrane. Plasmid DNA selectively adsorbs to membrane
- Plasmid DNA binds membrane under high salt conditions
- impurities washed away
- pure plasmid DNA eluted under low salt conditions
What is the most commonly used procedure to separate bacterial chromosomal DNA from plasmid DNA?
alkaline lysis
Anion exchange chromatography takes advantage of the fact that?
DNA is negatively charged
DNA quatification
- measure the absorbance of DNA at 260 and 280 nm.
- one A260 unit of double stranded DNA corresponds to 50 micrograms of DNA per ml
Formula to calculate DNA concentration
(a260)x(50ug/ml)x(dilution factor)=DNA con.
What are the ratios for pure DNA, contaminated DNA with protein or RNA?
DNA=1.8
RNA>1.8
Protein<1.8
When performing DNA agarose gel electrophoresis, DNA should be loaded in the gel closer to what end?
closer to the black (cathode) end
What can be determined by agarose gel electrophoresis?
Size and relative concentration
