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BIT 410 > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

biotechnology

A

the use of living organisms for industrial purposes

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2
Q

Genetic Engineering (molecular biotechnology)

A

the use of experimental techniques to produce DNA molecules containing new genes or new combination of genes

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3
Q

Overview of cloning project?

A

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

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4
Q

Plasmid

A

a circular piece of DNA, independent of the host chromosome, capable of replication

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5
Q

Vector

A

A DNA molecule (often plasmid) capable of replication in a host organism that is specially modified for propagating DNA sequences

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6
Q

Recombinant DNA

A

DNA created in vitro by joining together pieces of DNA that are not normally contiguous

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7
Q

Gene cloning

A

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

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8
Q

fusion protein

A

protein made by splicing two genes or gene sequences together, which when translated, produce a hybrid of chimeric protein

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9
Q

sub cloning

A

cloned DNA that is moved from one plasmid to another

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10
Q

Typical Steps in Traditional Gene Clothing

A

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

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11
Q

E. Coli plasmid (engineered and in nature)

A

AA

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12
Q

what are two types of commonly used plasmid vectors?

A

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.

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13
Q

Recombinant Proteins can be made using cloning vector?

A

FALSE

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14
Q

TorF: DNA can be inserted in an expression vector.

A

True

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15
Q

TorF: DNA is typically inserted into an engineered plasmid at the multiple cloning site.

A

TRUE

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16
Q

What are characteristics of a cloning vector?

A
  • High copy number
  • Cloning Site (multiple cloning site)-site where DNA is usually inserted
  • Origin of Replication
  • Selectable Marker
  • screenable marker
17
Q

What is a selectable marker vs a screenable marker

A

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.

18
Q

What is the purpose of selectable marker?

A
  • 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.
19
Q

WHY would you not want to lose the selectable marker?

A

because you would lose the ability to select and maintain selective pressure

20
Q

Characteristics of expression vector

A

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
21
Q

Alkaline lysis

A

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

22
Q

Denaturation

A

hydrogen bonding is disrupted and double stranded DNA is separated into single strands

23
Q

What are three similar steps that most plasmid purifications procedures start with?

A
  1. resuspend bacteria in buffer containing RNase
  2. Lyse bacterial membranes with SDS and denature DNA with NaOH
  3. Neutralize solution to reanneal plasmid DNA and precipitate out chromosomal DNA
24
Q

What are the two purification processes that we use in lab?

A

alkaline lysis

silica adsorption

25
Q

Anion Exchange chromatography

A
  • 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
26
Q

Silica adsorption

A

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
27
Q

What is the most commonly used procedure to separate bacterial chromosomal DNA from plasmid DNA?

A

alkaline lysis

28
Q

Anion exchange chromatography takes advantage of the fact that?

A

DNA is negatively charged

29
Q

DNA quatification

A
  • 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

30
Q

Formula to calculate DNA concentration

A

(a260)x(50ug/ml)x(dilution factor)=DNA con.

31
Q

What are the ratios for pure DNA, contaminated DNA with protein or RNA?

A

DNA=1.8
RNA>1.8
Protein<1.8

32
Q

When performing DNA agarose gel electrophoresis, DNA should be loaded in the gel closer to what end?

A

closer to the black (cathode) end

33
Q

What can be determined by agarose gel electrophoresis?

A

Size and relative concentration

BIT 410 (8 decks)

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