e.r. gauthier, ph.d.chmi 4226 f1 recombinant dna technology chmi 4226 e week 2 12 january 2009...

E.R. Gauthier, Ph.D. CHMI 4226 F 1

Recombinant DNA TechnologyCHMI 4226 E

Week 2

12 January 2009

Toolbox part 2.Plasmids, DNA cloning 101, DNA

sequencing


Plasmids• Plasmids:

– Extrachromosomal (episomal) circular DNA molecules– Minimal features:

• Origin of replication: allow the plasmid to replicate autonomously

• Antibiotic resistance gene: allow for selection for bacterial cells that have taken up the vector

• Multiple cloning site (MCS): a small region of the plasmid engineered to contain the cleavage sites for a limited number of restriction enzymes


Plasmids – origin of replication

• Origin of replication (Ori): small DNA sequences allowing the bacterial DNA polymerase to bind and initiate DNA replication;

• Several Ori can be found. ColE1 is the most frequently encountered;

• Some Ori allow the plasmid to replicate frequently (high copy number plasmids – up to 100 copies per cell); others allow only a low rate of replication initiation (low copy number plasmids – only a few copies per cell)


Plasmids – selection genes• Genes which encode for proteins affording resistance to a specific

antiibotic;• Most frequently encountered:

– Ampr: resistance to ampicillin– Kanr: resistance to kanamycin– Tetr: resistance to tetracyclin

• Bacteria possessing a plasmid with the Ampr gene will survive when plated onto a media containing ampicillin;


Plasmids – multiple cloning sites

• MCS: a limited region on the plasmid which has been engineered to contain unique cleavage sites for a selected number of restriction enzymes;

• RE cleavage sites in the MCS are not found anywhere else on the plasmid.


Plasmids – other features

• Promoters for viral RNA polymerases Sp6, T7 or T3– Allows for in-vitro trasncription experiments

• Origin of replication of phage F1:– Enables the production of single-stranded plasmids

• Genes encoding supressor tRNAs

• Reporter genes:– Allows for the easy identification of bacterial cells with desired

features– E.g.: Lac z (encodes -galactosidase)


Plasmids – SupF selection gene

• SupF: – Suppressor tRNA– Inserts an amino acid

(Glu) at the UAA stop codon

– Allows the survival of bacteria with a P3 episome : a plasmid containing the Ampr and Tetr genes both interrupted with a UAA stop codon.


Plasmids – pBR322


Plasmids - pBluescript


Plasmids – shuttle vectors

• Shuttle vectors can be used in at least two different organisms:– Bacteria (mandatory)– Yeast – Insects– plants– mammals


Plasmids – shuttle vectors

• Bacteria/Yeast shuttle vector;

• Leu2:– Selection marker for growth

in yeast– Encodes for a gene which

allows for the synthesis of the amino acid leucine in a yeast strain which cannot produce Leu on its own (auxotrophic)

– Yeast with this vector can be grown on media devoid fo leucine.


DNA cloning

• Why cloning DNA?– To produce greater amounts of YFG

– To characterise the properties of YFG• sequencing• Mutagenesis

– To express YFP in vitro or in a living organism• production of recombinant insulin: much better than insulin

purified from blood, which can be contaminated with viruses (hepatitis C, HIV) or other nice things (prions)


DNA cloning – basic steps• 1. cut DNA of interest and plasmid with appropriate

restriction enzyme

• 2. mix together and seal free ends with DNA ligase (ligation)

• 3. Insert DNA in bacteria (transformation)– Requires transformation-competant bacteria

• 4. Select antibiotic-resistant bacteria and search for cells having the recombinant plasmid.

• 5. Confirm cloning– restriction enzyme digest – DNA sequencing


DNA cloning – basic steps


DNA cloning

• Treating vector with alcaline phosphatase– reduces background of bacteria with plasmid not containing the

insert.

• Blunt-ends can be used:– a greater insert DNA/plasmid ratio must be used: 10/1 instead of

the usual 3/1– Much, much less efficient than cloning with protruding ends

• The ends of your DNA fragment can be modified with Klenow enzyme to accomodate the available RE sites in the vector (and vice-versa).

• Adaptors and linkers can be used to facilitate cloning.


Adaptors and linkers

Adaptors

Linkers


Assignment #2


Use of -gal- Bacteria used in genetic engineering express a portion of -gal missing the N-terminal 50 amino acids (called the omega [] fragment);

-Several vectors carry the missing N-terminal 50 amino acids ( fragment) of -gal in the MCS;

- Only the bacteria having both the and portion of -gal can express a functional enzyme ( complementation)


Use of -gal• If a DNA fragment is inserted

into the MCS of pBluescript, it interrupts the -gal fragment, making it inactive;

• SO: bacteria with a recombinant plasmid will not express a functional -gal, and will turn white upon staining wity X-gal;

• However, bacteria with a plasmid which does not contain the DNA fragment will produce the fragment, will have a functional -gal enzyme, and will turn blue upon staining with X-gal.


DNA cloning – plasmid purification from bacteria

Culture 1 bacterial colony into liquid media

Recover bacterial cells by centrifugation

1) Resuspend cells in glucose-based buffer

2) Lyse bacteria with SDS/NaOH buffer

3) Precipitate bacterial chromosome and proteins with acidic buffer and centrifuge

Extract supernatant with basic (pH 8) phenol - solubilizes contaminating proteins

Extract with chloroform – to get rid of the phenol

Precipitate plasmid DNA with ethanol

Dry pellet, resuspend in buffer, digest with restriction enzyme and analyse on agarose gel

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DNA cloning – identification of recombinant plasmids

• Three important tests have to be done:– Cut with RE to see if the

insert is of the correct size;

– Cut with RE to confirm the identity of the fragment (usually choose RE that cut inside the fragment)

– Sequence the insert.


DNA sequencing


DNA sequencing3’AGTCGCACTAGTGCATAG5’


DNA sequencing


What to do with the data?

• 1. identification (did I clone the piece of DNA I wanted)

• 2. Is it complete, or do did I clone only a portion of it (happens more often than you think…)?

• Are there any mutations (happens a lot…)?


BLAST search

• Blast: algorithm that allows you to quickly identify sequences that are similar to the DNA of interest;

• Blastp: similarity searches with amino acid sequences

• Blastn: similarity searches with nucleotide sequences


BLAST search

QUERY sequence(s)

BLAST database

BLAST program

BLAST results


BLAST search


BLAST search• E value: indicate the

odds that the hit is attributable to chance only.– The lower the E

value, the better the odds that it is a real match.

• G: link to Entrez Gene• U: link to sequence-

related info (expression profile, chromosomal location, orthologs)

• E: link to GEO (Gene Expression Omnibus): database of gene expression profiles.


BLAST search


Translation of sequencing data

• Allows you you determine if the sequence you isolated contains the entire open reading frame.– Open reading frame (ORF):

• the nucleotide sequence coding for a protein;

• Starts with a AUG codon, and stops with one of the 3 stop codons (UAA, UAG, UGA).


BLAST search


Blastp result


Assignment #3!

e.r. gauthier, ph.d.chmi 4226 f1 recombinant dna technology chmi 4226 e week 2 12 january 2009...

Documents

f2 plasmids plasmids

f6 plasmids

dna sequencing slide

f9 plasmids pbr322 slide

ampicillin slide

cell slide

stranded plasmids genes

cloning dna