Transcript Transforming E. coli with a Recombinant Plasmid Laboratory 5 Overview  Purpose:  Get the pARA-R plasmid containing the rfp gene into bacterial cells  Get.

Transforming E. coli with
a Recombinant Plasmid
Laboratory 5
Overview
 Purpose:
 Get the pARA-R plasmid containing the rfp gene into
bacterial cells
 Get those cells to express the rfp gene and make the
mutant fluorescent protein
Introduction
 Transformation
 Process of taking up foreign pieces of DNA
 In this case, a plasmid
 Usually happens through conjugation
 Not very common in mature!
 Can occur under experimental conditions (1 cell in a thousand!)
 Advantages include antibiotic resistance
 All cells that undergo binary fission after insertion of plasmid
will possess it:
Plasmid DNA Insertion
Transgenic Colony Allowed to Grow
Introduction
 Transformation efficiency:
 Size of plasmid
 Large
 Less likely to take up plasmid
 Must pass through plasma membrane and cell wall
 Small
 More likely to pass through
 Shape of plasmid
 Supercoiled easiest to pass through
 Nicked-circle or mulitimer harder to get through
 Most likely your tubes will contain
Introduction
 What are competent cells?!?!
 Cells ready to receive plasmids through lab procedures
 Soaked in calcium chloride
 PM and DNA negatively charged
 Calcium ions (Ca++) neutralize charges to allow for plasmid to
pass through PM
 Need to “heat shock” cells
 Creates pressure differences
 Cold then hot
 Then feed and recover!!
Bruce Wallace
Preparing competent cells for transformation
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
Transforming Escherichia coli with pARA-R
Bruce Wallace
Competent Cells
pARA-R
Recombinant Plasmids
Bruce Wallace
Transforming Escherichia coli with pARA-R
Lipid bilayer
(inner)
Peptidoglycan
layer
Adhesion zone
Lipid bilayer
(outer)
Calcium ions
pARA-R
Introduction
 Spread cells on various plates (3)
 LB plate
 Only bacterial food
 LB/amp plate
 Contains ampicillin
 Antibiotic prevents bacteria from forming CW
 Cells that contain ampr produces protein that destroys
ampicillin
 Cells will grow!
 LB/amp/ara plate
 Contains arabinose
 Needed to express the rfp gene
 If take up plasmid, this helps with transcription of gene
Materials
 Reagents and Cultures
 Equipment and Supplies
 pARA-R tube
 P-20 micropipette and tips
 100 µL competent cells
(LMG)
 P-200 micropipette and tips
 350 µL LB broth (sterile)
 1 pack sterile spreaders
 Crushed ice in Styrofoam
cup
 Plastic microfuge tube rack
 Sterile agar plates
 LB
 LB/amp
 LB/amp/ara
 42oC water bath
 1.5 mL microfuge tubes
 Marking pens
 Disinfectant spray
 Cybersafe
What will you need to do?
 Discuss proper aseptic techniques
 Protect themselves
 No contamination!
 Assign tasks….
 Turn on water bath the day before to check
temperature
What will you need to do?
 KEEP COMPETENT CELLS FROZEN until transformation day
 Day of lab:
 Take out number of tubes need for transformation
 Each group will need 100 µL of cells
 Each tube contains @ 500 µL of cells
 Place tubes in wet ice to defrost
 Will take about 10-15 minutes
 Resuspend cells prior to aliquoting
 Gently pump in and out with P-200 pipette
 Return any unused tubes with cells immediately to Styrofoam
chest and place into freezer
What will you need to do?
 Cells need to be in contact with
ice or water during exercise
 Push tubes to bottom of foam rack
 Have cell-contaminated waste
bag
 Deposit tips, tubes, and spreaders
that have come in contact with
bacteria
 Bag will be autoclaved after use
Methods….diluted version
 2 clean microfuge tubes – KEEP PIPETTE TIPS SEPARATE!!!!
 P+
 P-
 Label BOTTOM of plates (agar side) near edges
 After step 12, let rest for a few minutes at room temperature
 Step 14d
 Clamshell opening
 GLIDE spreader, do not dig
 Be sure to invert the plates when incubate
 Decrease condensation and skewed results
Predictions……
P+
P-
LB
P-
P+
LB/amp
P+
LB/amp/ara
Predictions……
P+
P-
+
+
LB
P-
-
P+
+
LB/amp
P+
+
LB/amp/ara
Conclusions
 2 tubes
 P+ contains E. coli and plasmid
 P- contains only E. coli
Bruce Wallace
Growth of transformed bacteria on various plates
P+ plates
LB
LB/amp
P- plates
No growth
LB
LB/amp
LB/amp/ara