Experiment 2 - Isolation, cloning and transformation of plasmid DNA

1. chromosome or
2. extra chromosomal piece of DNA ie\ phage or plasmid

1 *cloning, sequencing and expressing* foreign
genes in E. coli

1c. A DNA squence with MCSintroduced into this region

1. isolate plasmid DNA – pKan and pUC18

1a. The pUC plasmids contain:
– [pBR322] ampicillin resistance gene [the beta-lactamase (bla) gene] – origin of DNA replication

1b. plasmid pKan contains a kanamycin resistance gene.

2. When introduced into a suitable E. coli DH5a host strain carrying a lacZ portion that is able to
complement the portion on the plasmid, plasmid ligate to that portion of the lacZ gene as well as MCS –> transformed strain

X-Gal [(5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside)] is an inert chromogeni substrate –> cells with LacZ are able to make beta-galactosidase –> hydrolyzes X-Gal on plates into [colorless galactose and 4- chloro-3-brom-indigo, forming an] intense blue precipitate.

4. Cloningligating restriction DNA fragments (kanamycin resistance gene) into the Multiple Cloning Site of pUC18 vector = recombinant plasmid –> inactivates the lacZ gene, therefore cells of E.coli strain DH5a transformed with these new recombinant plasmids can not make betagalactosidase on selective media (x-gal)
and therefore produce white colonies on the agar plates –> indicate the presence of the kanamycin gene inserted into the pUC18 vector.

6. These colonies will then be streaked onto kanamycin/ampicillin (or carbenicillin) plates
to verify the presence of the kan and amp gene in the vector. E.coli DH5a colonies with resistant to both will be selected

confirm that plasmid DNA was extracted

restriction enzymes – Hindii, bamhii

1. a semi-synthetic ampicillin analog
2. b-lactamase (bla) gene also confer resistance to carbenicillin
– more stable than ampicillin ie\ less likely to breakdown in presence of beta-lactaases
– reduce the growth of satellite colonines on plates during long-term incubations

An autonomous, self-replicating, extrachromosomal DNA mole

Incorporating a DNA molecule into a chromosomal site or a cloning vector

A population of cells that carry a cloning vehicle with the same DNA insert

The *uptake of extrachromosomal DNA* in a bacterium or yeast cell in
which the DNA often *changes the phenotype* of the recipient organism

A cell which has *taken up extrachromosomal DNA* and is expressing a
change in *phenotype*

Cells which have been treated by chemical or physical means in order
to be sensitive to foreign DNA

bluewhite selection technique to select colonies with a successful insert (kanamycin resistance gene)

Plate 100 μL from tube 3 onto 3 LB/carb/kan plates

Re-suspension

Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall.

EDTA or Tris
– chelates (carries) divalent cations in the solution preventing DNases from damaging the plasmid
– destabilizing the cell wall

Glucose
– maintains the osmotic pressure so the cells don’t burst, also pH

– RNase A degrade cellular RNA when the cells are lysed. –> we want to keep the DNA plasmid

SDS
– detergent
– break down cell membrane
-denatures cell proteins –> help w later separation of proteins from plasmid

NaOH
[break down cell wall] alkaline lysisdenaturation – disrupts H-bonds between DNA –> double-stranded plasmid DNA to single stranded DNA (ssDNA)

Neutralization

Solution becomes more basic –> *hydrogen bonding re-established* –> *ssDNA re-nature to dsDNA* ie\ circular plasmid DNA re-nature

bacerial genomic DNA (gDNA) is very long relative to plasmid DNA –> plasmid DNA reforms much easier and faster than gDNA –> selective part.

impossible to properly anneal those huge gDNA stretches.

–> EXPERIMENTAL ERROR: This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

separated from the plasmid DNA solution by centrifugation.

*double-stranded plasmid* *dissolve* easily *in solution*,

single stranded DNA + SDS + denatured cellular proteins stick together through hydrophobic interactions and precipitate

plasmid DNA separated from the majority of the cell debris

solution highly contaminated – salt, EDTA, RNase, residual cellular proteins and debris –> not much use, need to purify more

organic solvent

concentrate and precipitate the plasmid DNA from the supernatant to be isolated

clean up the solution

nucleic acid operations

Purification of DNARNA

E. coli

Remove 5′ phosphates from the ends of DNA vector that have been cut with a RE

5’P-DNA –> 5’OH-DNA + Pi

prevents self-ligation (recirculization) of the vector in subsequent ligation reactions –> facilitates ligation of other DNA fragments into the vector (e.g. subcloning).

transform a strain of E. coli DH5a to be resistant to both ampicillin and kanamycin by creating recombinant pUC18 plasmid by restriction digestion and cloning

confirm that plasmid DNA was extracted

pKAN – 4194 bp
pUC 18 – 2686 bp
pKAN will appear above

1. different plasmid topologies =
plasmid conformations

6. Nicked
linear
supercoiled (thicc)
circular, single-stranded

3. structure deviates from the theoretical “circular” plasmid molecule

4a. treated lysate too vigourously
4b. digestion of the sample with EcoRI
4c. smear in contrast to linear band after digestion of multimeric plasmid forms

5. supercoiled

1. fused products of several plasmids recombined together
1a.several times larger than the individual plasmid –> run very slowly in agarose regardless of their conformation

1a. interested in this one
1b. (pUC) lower band compares to circular (also pUC) (migrates faster than predicted)
1c. (pTZ19) multimeric forms of supercoiled plasmid DNA
– a tiny bit above the pUC supercoiled
– not genomic DNA
– linearization using restriction digestion, genomic DNA –> single defined band with the size of the linearized (plasmid monomer) form

3a. denatured supercoiled DNA
– very weak band below pUC supercoiled

2a. native conformation found in vivo
2b. extra twists in the double helix strand.*

1c. relaxed circular plasmid
1a. during replication, cell topoisomerases nick on strand of the DNA helix and relax the superhelical tension, allowing polymerases to gain access to the DNA
1b. slowest migrating form in an agarose gel

1a. (pTZ19) linearized form
– same with (pUC) open circular form
1b. after restriction digestion with EcoRI –> DNA helixplasmid is cut in both strands at the same place
1c. nuclease contamination or harsh treatment during purification

1a. (pUC) open circular form
– upper band compares to supercoiled (pUC)
– ds DNA
– nicked in one of the strands to released supercoils

1b. harsh alkaline lysis step bc incubated for too long

1c. DNA become permanently denatured resulting in single stranded closed circles