Download Lab 10 Study Guide

S. Badran
BIO 107 Lab # 10: Summary & Study guide for Day I, II & III Labs
You transformed E. coli with pGLO plasmid that contains GFP gene, Arabinose C gene and ampicillin resistance gene. The
transformation was done using sterile technique & heat shock to obtain competent cells that can take up the pGLO
plasmid. Cells were incubated at 37 oC overnight, and transformation data were analyzed and evaluated to calculate
transformation efficiency. Transformed cells grew on LB/AMP/Arabinose medium and glowed green when exposed to UV
light, since they expressed the green fluorescent protein, ampicillin resistance enzyme and arabinose protein (which
activates GFP gene). Transformed cells were then placed in sterile culture medium containing LB/Ampicillin/Arabinose and
grown at 37 oC overnight in order to stimulate the production of green fluorescent protein. Hydrophobic Interaction
Chromatography (HIC) was used to separate green fluorescent protein from the remaining cell components and purify it.
Green fluorescent protein was easily identified in the final chromatography fraction by using UV light and observing the
protein glow green.
Part I: Bacterial Transformation using heat shock
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Review using these videos:
Bacterial transformation using electroporation:
http://passel.unl.edu/pages/index.php?allanims=1
Bacterial transformation using heat shock:
http://www.dnai.org/lesson/go/59/8446
Early genetic engineering experiment:
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
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Genetic transformation is the process by which cells acquire & express new genes.
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Transformation can occur in nature, when cells pick up naked DNA that leaks out of dead cells. In the lab, cells can be
induced to pick up DNA or plasmids, if they lack the mechanism necessary for DNA uptake, which was the case for the
E.coli strain we used
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You used sterile technique & heat shock to induce E.coli to take up the pGLO plasmid
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The heat shock procedure used the following:
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relatively high concentration of calcium ions in order to neutralize the negative charge of DNA so it can pass the
negative lipid bi-layer
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Heat shocking cells by suddenly changing temperature from ice to 42 oC back to ice; this makes the membrane
more fluid & permeable in order to create temporary pores, through which the plasmid can pass
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Heat shock yielded competent cells: cells in a state that allows them to be transformed.
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A recovery period was required after heat shock & before plating competent cells out.
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Recovery was done by incubating cells in LB at 37 oC to give them a chance to replicate the acquired plasmid,
transcribe & translate its genes, so they are ready for cell division. This also prevents competent cells from being
inhibited or killed immediately when plated out on LB/Amp medium.
Control & experimental group: sterile technique and heat shock were applied to 2 identical samples; except that one
contained pGLO plasmid (P+), while the other sample did not (P -). The sample that did not contain plasmid was treated
equally & served as negative control to show how competent E.coli behave on different growth media when they lack
pGLO plasmid.
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Plating out cells: competent cells from the experimental & control group were plated out on two types of growth medium:
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LB: Luria broth, which contained minerals, peptides vitamins etc. All E. coli grow well on LB
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LB/Amp/Arabinose: ampicillin & arabinose were added to select for transformed cells only. Ampicillin killed all nontransformed cells (this E.coli strain is not naturally resistant to ampicillin). Arabinose C is a small sugar that
activated the Arabinose C gene, so that it is expressed into an activator protein. This activator was needed for the
expression of green fluorescent gene
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Plasmids are double stranded circular DNA that exist in addition to the single chromosome in bacteria. They carry genes
that may be beneficial under environmental stress allowing cells to adapt very quickly to a changing environment. This
causes evolution to occur over very short period of time.
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Features of the pGLO plasmid:
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pGLO carries a GFP gene, which codes for green fluorescent protein. It was cut out of the genome of the jelly fish
Aequorea victoria, which glows green in deep sea. GFP gene was then cloned into this plasmid. Cutting and
pasting is done using restriction enzymes that produce compatible sticky ends on the plasmid & GFP gene.
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pGLO carries an Arabinose C gene, which expresses an activator protein. This activator acts as switch to turn on
the GFP gene, so it gets expressed into GFP protein. This allows transformed cells to glow green when grown on
LB containing arabinose
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pGLO is an R plasmid: it carries an ampicillin resistance gene that codes for an enzyme called beta lactamase,
which breaks down ampicillin. This allows transformed E.coli to grow on LB/Amp medium, while non transformed
E.coli are inhibited by ampicillin (this E.coli strain is NOT originally ampicillin resistant)
Part II: Data Analysis & Transformation Efficiency
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Review using the handouts given during the lab session
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Expected data should yield the following:
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LB medium: will contain a “lawn” of colonies (lots of growth) for both samples; P + and P –
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LB/Amp/Arabinose will show green glowing colonies for the P + sample only. The number of colonies will vary
based on transformation efficiency.
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–
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Reason: Arabinose will activate Arabinose C gene, which expresses an activator protein. This activator
acts as switch to turn on the GFP gene, so it gets expressed into GFP protein. Ampicillin resistance
gene will code for beta lactamase, which breaks down ampicillin, allowing only transformed E.coil to
grow, while non transformed cells are inhibited by ampicillin.
LB/Amp/Arabinose should not show any colonies growing from the P – sample.
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Reason: all cells can use LB to grow
Reason: Non transformed cells are inhibited by ampicillin.
Some actual data deviated from expected data due to a number of reasons:
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Transformation may not have taken place (no green colonies on LB/Amp/Arabinose that contained P + sample)
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Contamination may have occurred (ex. if growth is observed on LB/Amp/Arabinose plates that contained P –
sample
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Most cells may have been killed during transformation procedure, if very little or no growth is observed on most
plates.
Transformation efficiency: represents the number of transformed colonies per mass of plasmid (in ug)
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too much DNA inhibits transformation, so about 5 to 10 nanograms are used.
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Transformation efficiency ranges from 104 to 107 cells per microgram DNA.
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To calculate transformation efficiency, refer to the problems discussed in class in addition to your own data and
calculations. Below is an example:
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Step 1: Determine mass of the plasmid added to the first transformation tube (+ Tube):
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0.005µg/µl x 10µl = 0.05 µg plasmid
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Step 2: Determine the concentration of plasmid in the reaction tube before plating out:
_____________0.05 µg _______________
= 0.0001 µg plasmid/µl
(250µl LB broth + 10µl Plasmid + 200µl CaCl2)
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Step 3: Find mass of plasmid spread on agar plate
0.0001 µg plasmid/µl X 100µl = 0.01µg pAmp
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Transformation efficiency:
50 transformed colonies = 5000 colonies per microgram of plasmid
0.01µg pAmp
Part III: Purification of GFP by Hydrophobic Interaction Chromatography (HIC)
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Chromatography is an analytical method that separates a mixture into its molecular components. HIC was used to
separate the cloned GFP from other cell culture components
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HIC separates the desired protein based on its affinity to a ligand (molecule with specific shape) that is attached to
beads in a column. HIC column contained beads that are attached to a hydrophobic ligand. GFP is very hydrophobic so
it strongly binds to the beads of HIC column. Other, less hydrophobic (more hydrophilic) proteins & cell components will
pass through the column
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Buffers of different strengths were added to wash all undesirable components off HIC column without affecting the bond
between HIC column & GFP. Different “flow through samples” or fractions were collected. Finally, a buffer was added
to force the GFP to separate from HIC The fraction containing GFP was identified using UV light (glows green)
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Transformed cells were placed in sterile culture medium containing LB/Ampicillin/Arabinose and grown at 37 oC
overnight in order to stimulate the production of green fluorescent protein
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Lysozyme was added to culture to lyse cells (break open cells); then cells were incubated in -20 to – 80 oC freezer.
This allowed ice crystals to form, which helped disrupt cell walls, membranes, and intracellular vesicles.
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Centrifugation following freezing & thawing caused larger pieces of cellular debris to form a pellet, while leaving the
protein of interest (GFP) in the supernatant (liquid).
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The supernatant containing GFP is prepared for HIC by adding binding buffer. Binding buffer contained the highest
concentration of ammonium salt. It caused GFP to undergo conformational changes that exposed its hydrophobic
functional groups, so they moved to the exterior of the molecule. This in turn allowed GFP to strongly bind to HIC
column, when supernatant/binding buffer mix was poured over the column.
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The following buffers were used in this order to purify GFP from a HIC column:
o Equilibrium Buffer: packed the beads tightly on HIC column before use
o Wash buffer: removed any cell debris & undesired proteins. It has a lower salt (ammonium sulfate)
concentration than binding buffer. Low salt concentration begins to reverse 3-D changes of GFP; GFP is still
bound to column, but less strongly than before
o TE buffer: it contains no salt, allowing GFP to return to its normal 3-D shape. Once this occurs, the GFP can no
longer interact with the hydrophobic resin.
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In the biotechnology industry: production, separation & purification of therapeutic proteins are essential for marketing.
Separation & purification can be complicated & very expensive, but the FDA allows the marketing of functional
recombinant proteins with high purity only
Study Guidelines:
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Review the concepts and methods involved in this lab using the lab manual, PowerPoint presentation,
videos/animations and transformation handouts.
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Review this document to summarize and check your understanding and to review the new terms.
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Make sure you know how to analyze & interpret your transformation data and any data obtained from other hypothetical
transformation experiments; in addition to calculating transformation efficiency. You will need to generate possible
explanations for unexpected transformation data
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Make sure you know the purpose of all the major steps and components of bacterial transformation and protein
purification by HIC.
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Review the guidelines for micropippetting and pouring/running gels very carefully to make sure you know what mistakes
to avoid.
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Make sure you can correctly interpret the micropipette counter into volume in ul (3 vertical numbers shown in the
window of the micropipette)
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Review metric unit conversions