1
11a Plasmid
3723- F02
Biochemistry 3723Lecture 11: DNA Technology # 1 Nov. 11, 2002
Objectives
1.Introduce the techniques of recombinant DNA technology
2.Understand use and limitations of recombinant DNA technology
I. Process of Cloning--see flow chart next page--
A.Purpose--to isolate, sequence, manipulate well defined pieces of DNA
B.Preparation of genomic DNA (done ahead by Judy)--
1.Isolate DNA-free from proteins--usually phenol extraction, ethanol precipitation
a.source?
b.ours is "mystery" DNA--part of your assignment is to determine source
2.Cut into managable sized pieces--
a.Restriction enzymes
b."6 cutter" ~ 4 kb size
3.Can determine size using agarose gel electrophoresis--more later.
C.Vector: we use plasmid pBluescript®I SK+ plasmid
1.plasmids as vectors for carrying foreign DNA-- for DNA up to ~5kb
a.independent replication (not tied to bacterial chromosome)
--ori plus cis acting controlling elements = replicon
-- if “relaxed” can make many (hundreds)/cell
b.small size (<10 kb; 2-5 kb ideal) pBluescript is 2.96 kb
c.selectable marker: allows ONLY cells with plasmid to grow under test conditions--usually antibiotic resistance gene
d.MCS: multiple cloning site-unique restriction sites engineered into plasmid: one site/plasmid in screening element
e.screening element--usually by “insertional inactivation” Now almost universally -galactosidase gene More later
f.promoters: flanking MCS (examples, T3 and T7).
i.Allow controlled transcription to mRNA--also as primer site for DNA sequencing
ii.Expression vectors have especially strong promoters
g.ssDNA propagation useful
2.Other vectors--mention only
a.Phage lambda--engineered to allow insertion of large pieces of DNA (10-20 kb)
b.Cosmids: Combined lambda and plasmid (have ori, can package as virus) up to 45 kb
c.YACs: up to 500 kb, but must work in yeast.
d.BACs; also for large pieces of DNA
C.Joining "passenger" DNA to vector by restriction and ligation
1.Restriction of DNA: a little about restriction enzymes (endonucleases).
a.There are three types. Most useful for recombinant DNA technology is
Type II--cuts at recognition site, no ATP required, no methylase activity
b.Recognition site: palendromic sequence
EcoRI 5'GAATTC3' G +5'AATTC
3'CTTAAG5' CTTAA5'G
SacI5'GAGCTC3' GAGCT3' +C
3'CTCGAG5'C3'TCGAG
SmaI5'CCCGGG3'CCC+GGG
3'GGGCCC5'GGGCCC
c.Frequency of cutting: 1/4n; n = 4 1/256;n = 6 1/4096
d.Ends:
i.sticky: 5' overhang (EcoRI); 3' overhang (SacI)
ii.blunt end (SmaICCC/GGG)
2.Restriction using EcoRI or EcoRI + SacI
a.When cut genomic DNA with EcoRI only get average size of 4 kb. When cut with two "6 cutters" average size would be smaller.
b.When cut pBluescript with EcoRI, get only one cut at well-defined location within MCS. When cut with both EcoRI and SacI, get one large piece and one small piece (from SacI to EcoRI) of ≈ 50 bp
c.In theory should inactivate enzymes by heating after restriction, or re-purify the DNA by phenol extraction. In practice is often unnecessary
d. Some more on restriction
i.methylation--how bacteria keep from chewing up own DNA?
ii.Single vs. double digests (we're doing double digests)
iii.Cost: $ 0.005-1.00/unit. Use 1-10 units/tube
iv.Glycerol for stability in storage, must be diluted out for activity
v. Instability of enzymes: Keep cold
vi.Star activity and buffers.
3.Ligate the genomic DNA into the plasmid (did Monday)
a.Enzyme: T4 DNA ligase (from bacteriophage T4). Requirements: Mg++and ATP
b.Temperature--why at 12°C?--activity vs. specificity (stability of bps)
c.There is a kit now available that allows ligation in 5 minutes at room temp. Go figure!
D.Today: Transformation: Getting DNA into bacterial cells
1.Requires competent cells: What is that? The fine art of making competent cells. We bought them.
2.Under appropriate conditions, Ca++ treatment and sudden temperature changes, competent cells take up naked DNA (closed circle best)
3.Cells: DH5(recA-) E. coli strain with special characterisitics:
a.Contain no plasmid
b.no functional -galactosidase
c.no growth in presence of ampicllin
d.highly efficient competent cells
e. dam- and dcm- (no methylases)
E.Selection and Screening:
1.Define difference between them
a.Selection--only selected cells can grow under chosen conditions (+ Ampicillin means only plasmid-containing cells grow)
b.Screening--means of choosing those clones that grow which have desired characteristic (passenger DNA insert)--Blue /White screen
2. Plate on TAXI plates
a.T = thiamine; required for growth of thiamine auxitroph (DH5
b.A = ampicillin--selection marker; DH5 won't grow, but DH5transformed with plasmid DNA will grow (plasmid has ampr gene)
c.X = X-gal (5-bromo-4-chloro-3-indoyl--D-galactose): Substrate for -galactosidase; Turns colonies blue upon hydrolysis. But won't induce lac operon.
d.I = IPTG (isopropyl --D-thiogalactose): “Gratuitous” inducer of lac operon, but not metabolized by -galactosidase.
3.Results:
a.Only cells transformed with plasmids will grow. This is selection
b.IPTG induces lac operon but is not metabolized.
c.X-gal is metabolized to give blue color (same as in alkaline phosphatase assay for western blots but only in cells with an intact -galactosidase region in the plasmid.
d.Cells containing plasmid w/o insert have functional -galactosidase region: X-gal Gal + BCI-OH (blue); colonies are BLUE
Cells containing plasmid w/ passenger DNA inserted at MCS have interrupted gene and no functional -galactosidase: X-gal no reaction; colonies remain WHITE. This is BLUE/WHITE screening.
e.There are always exceptions (my "fish-eyes").
f.Feeder colonies--cells without plasmid that grow after ampicillin degraded by plasmid containing cells.
F.Procedure for today
1.Transformation
a.Use 2 concentrations of ligated DNA--Why? Just to increase chance of easy to score (+) response. More later.
b.Incubate DNA with compenent cells 30 min. on ice; heat shock @ 42° C for 40 sec, back on ice 2 min.
c.Recover at 37°C, ~1 hr to make sure DNA is completely in and being expressed. If not expressing plasmid DNA, cells may not grow, because we then grow them in medium containing ampicillin. Cells depend on plasmid expression to inactivate ampicllin and allow growth.
2.After 1 hour incubation: Plating--spread cells on plates: Demo in lab
3.Incubate overnight @37° C upside down (why?)
G.Procedure for TOMORROW--we will not do for you
1.Pick white colonies that are cleanly separated from blue ones. into broth culture of LB + Ampicillin. Only cells containing plasmid can grow. Ideally would have streaked out in between to insure that have single colony.
2.Grow overnight. Will do mini-prep on Wednesday
H.Specifics you should be aware of
1.Restriction of genomic DNA is incomplete. Means not every SacI or EcoRI site has been cut.
2.This presents multiple possibilities when it comes to inserts.
a.Sac-Sac, Eco-Eco as well as Sac-Eco are possibilities
b.Inserts with internal Sac or Eco sites also possible.