ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides the ability for a cell or organism to respond to a constantly changing external environment. The specific constellation of proteins required for optimal function and growth varies with cellular age and environmental context. Thus, protein production is carefully regulated by multiple mechanisms that modulate both transcriptional and translational pathways. Control of transcription initiation by RNA polymerase is a predominant mechanism for regulating expression of specific proteins, presumably because it provides maximal conservation of energy for the cell. We can often observe the consequence of differential transcription due to the presence or absence of particular proteins or the growth in particular environments. Control can also occur at translation; the mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at the level of protein function; the protein is inactive, or activity is not observed due to the lack of the substrate. In this lab we will observe differential expression of two different genes encoded on plasmids. We will analyze transcriptional activity, translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early work on the plasmid series had been conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. - pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of recombinant.

1. ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest can

2. be distinguished from cells with the plasmid but without the gene of interest, just by looking
assaying for the functional -galactosidase. The enzyme's typical substrate is lactose, but it will
bind and cleave a variety of analogues. Enzymatic activity (and therefore expression) is
monitored by the color of the colonies. The analogue ONPG when cleaved produces a yellow
color, X-gal produces a blue color. - The multiple cloning site (MCS) region is split into codons
6-7 of the lacZ gene, providing for many restriction endonucleases restriction sites. If DNA is
inserted is ligated into the MCS, the LacZ gene coding sequence is disrupted, and no functional
protein is produced. This is noted by the color of the colonies (recombinant white, non-
recombinant yellow or blue depending on substrate in the media)
The pGLQ plasmid is an engineered plasmid used in biotechnology as a vector for creating
genetically modified organisms. The plasmid contains several reporter genes, most notably the
green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the
jelly fish Aequorea victoxia. Because it shares a bidirectional promoter with a gene for
metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the
transgenic organism express its fluorescence under UV light. GFP can be induced in bacteria
containing the pGLQ plasmid by growing them on +arabinose plates. pGLQ is made by Bio-Rad
Laboratories. - pGLQ contains an origin of replication and is maintained in high copy number -
bla is the gene that codes for -lactamase and is the selectable drug-resistant marker. It is under
the control of its own promoter. The expression of the gene results in a protein that cleaves the -
lactam ring of ampicillin (and penicillin) allowing for the growth of the bacteria in the presence
of the antibiotic. E. coli are normally highly sensitive to ampicillin. - The GFP is a protein that
causes fluorescence in the presence of UV light. The protein was originally cloned from an
aquatic species. The protein is harmless to the bacteria, but can serve as a useful marker. In the
pGLQ plasmid the GFP is linked to the arabinose PBAD promoter. This is a strong promoter and
will cause RNA polymerase to make a large number of copies of mRNA from this gene (and
therefore, a lot of GFP protein). - The arabinose PBAD promoter is regulated by the protein
coded for by the axaC gene which has its own promoter. - In the absence of the sugar arabinose,
the araC protein binds to the PBAD promoter and prevents transcription of GFP. - In the
presence of arabinose in solution, the araC protein binds the arabinose, and this results in a
conformational change to the araC protein, the result of which is that it now instructs RNA
polymerase to make many copies of the GFP mRNA (and thus, a lot of GFP protein)
I. LacZ expression - E. coli transformed with pUC 19 (the plate contains amp and Xgal/PTG)
was incubated for 24 hours at 37C. - E. coli transformed with pUC 19+insert DNA (the plate
contains amp and Xgal/IPTG) was incubated for 24 hours at 37C. II GFP expression - E. coli

3. transformed with pGLQ grown on a plate containing amp was incubated for 24 hours at 37C. - E.
coli transformed with pGLQ grown on a plate containing amp/ara was incubated for 24 hours at
37C. III. RNA isolation and analysis mRNA was isolated from the bacteria from each of the
plates. The mRNA was converted to DNA with RT enzyme. You will perform a microarray to
assay LacZ gene transcription (2 different conditions) and GFP gene expression (2 different
conditions). An array is an orderly arrangement of samples where matching of known and
unknown DNA samples is done based on base pairing rules. An array experiment makes use of
common assay systems such as microplates or standard blotting membranes. Thousands of
spotted samples known as probes (with known identity) are immobilized on a solid support (a
microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or
oligonucleotides. These are used to determine complementary binding of the unknown sequences
thus allowing parallel analysis for gene expression and gene discovery. An experiment with a
single DNA chip can provide information on thousands of genes simultaneously. An orderly
arrangement of the probes on the support is important as the location of each spot on the array is
used for the identification of a gene. The sample has genes from both the normal (in this assay
green) as well as experimental conditions (in this assay red). Spots with more intensity are
obtained for experimental gene if the gene is over expressed in this condition. This expression
pattern is then compared to the expression pattern of the gene in the control growth conditions.
You will be testing the mRNA from the 4 plates for the expression of two different genes: lacz
and gfp. Your analysis will also include 4 controls. Protocol-card preparation 1. Add 5l of
equilibration buffer to each spot on the microarray card. 2. Incubate at 37C until the card is dry.
3. Add 5l of the blocker to your cDNA samples (4 assay controls and the cDNA from the E.
coli.). Mix up and down with the pipetter. 4. Add 5l of the sample to the appropriate spot on the
card. 5. Repeat step 3-4 for the remaining samples (7 more times) 6. Incubate at 37C for 5
minutes or until the card is dry. 7. Add 5l of the hybridization buffer to each spot. 8. Incubate at
37C for 5 minutes or until the card is dry. 9. Visualize with the handheld UV light. 10. Record
your results (photo)
IV Protein isolation and analysis Protein was isolated from the bacteria from each of the plates.
You will perform a protein gel analysis to assay LacZ protein size and quantity (2 different
conditions) and GFP size and quantity (2 different conditions). Protein Gel Assay When proteins
are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less
resistance from the gel matrix. Other influences on the rate of migration through the gel matrix
include the structure and charge of the proteins. Sodium dodecyl sulfate (SDS, also known as
sodium lauryl sulfate) is a detergent with a strong protein-denaturing effect and binds to the
protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that

4. cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with
negative charge proportional to the polypeptide chain length. SDS largely eliminates the
influence of the structure and charge, and proteins are separated solely based on polypeptide
chain length. You will compare the protein gel assay from the 4 different conditions to assay for
the presence of the GFP and LacZ proteins. Protein gel assay 1. Load 20l of the protein marker 2.
Load 20 of the samples (GFP -Ara and +Ara, LacZ parent, LacZ-insert) 3. Electrophorese that
samples at 120V for 1 hour 4. Submerge the gel in fixative and add 1 protein iostastain card 5.
Wrap with saran wrap and incubate 3060 minutes at 37C 6. Record (photo) your results
III. RNA isolation and analvsis 1. Describe the transcription of LacZ and GFP in the two
different growth conditions