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