1) DksA is a transcription factor in E. coli that plays an important role in stress response. It binds directly to RNA polymerase, not DNA.
2) The dksA gene has three temporal promoters that are expressed at different growth phases. One promoter, P3, is located within another gene, sfsA, and is followed by a putative open reading frame (ORF) of unknown function.
3) The goal was to determine if this ORF encodes a polypeptide co-expressed with DksA during stationary phase. However, experiments failed to detect expression of the putative ORF polypeptide, even with various techniques. This suggests the ORF is not translated into a
Reverse transcription of RNA is a process whereby RNA, typically messenger RNA is converted into complimentary DNA. The process was discovered by Howard Temin and John Baltimore when they observed that certain RNA viruses could revert to DNA. This was an important discovery that led to the discovery of enzymes classified as reverse transcriptases. Today Reverse Transcription is routinely applied in molecular biology laboratories to obtain the stable cDNA version of RNA for downstream analysis.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Reverse transcription of RNA is a process whereby RNA, typically messenger RNA is converted into complimentary DNA. The process was discovered by Howard Temin and John Baltimore when they observed that certain RNA viruses could revert to DNA. This was an important discovery that led to the discovery of enzymes classified as reverse transcriptases. Today Reverse Transcription is routinely applied in molecular biology laboratories to obtain the stable cDNA version of RNA for downstream analysis.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Whiteboard2Boardroom collaborates with more than 21 research institutions, hospitals and corporation to move technologies out of research labs into the marketplace.
Directions to "An Illustrated DNA Tale" a comical guide to protein synthesis. Students design a comic strip using non-science terms to depict a "tale" paralleling protein synthesis.
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency
Scientific research paper summary for class on molecular genetics. Original research on the development and use of long non-coding RNA in biological systems like CRISPR and the origin of that mechanism.
Proposed kinetic improvements to a Zika Biosensor developed by the Collins Lab at MIT that would reduce time of detection by 80% with a cost increase of only $0.01 per reaction.
Whiteboard2Boardroom collaborates with more than 21 research institutions, hospitals and corporation to move technologies out of research labs into the marketplace.
Directions to "An Illustrated DNA Tale" a comical guide to protein synthesis. Students design a comic strip using non-science terms to depict a "tale" paralleling protein synthesis.
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency
Scientific research paper summary for class on molecular genetics. Original research on the development and use of long non-coding RNA in biological systems like CRISPR and the origin of that mechanism.
Proposed kinetic improvements to a Zika Biosensor developed by the Collins Lab at MIT that would reduce time of detection by 80% with a cost increase of only $0.01 per reaction.
A CRISPR/Cas9, works like a biological version of a word-processing programme’s “find and replace”. Its simplicity and extremely low cost of implementation is the reason to use. How Cas 9 is activated and its mechanism (DNA binding and cleavage), it's regulation and application in human disease therapy, new drug screening, agriculture and biofuel etc.
INTRODUCTION
HISTORY
WHAT IS TRANSCRIPTION
PROKARYOTIC TRANSCRIPTION
STEPS OF TRANSCRIPTION
HOW TRANSCRIPTION OCCURS
PROCESS OF TRANSCRIPTION
Initiation
Elongation
Termination
CONCLUSION
REFRENCES
RNA polymerase and transcription factorKAUSHAL SAHU
INTRODUCTION
WHAT IS TRANSCRIPTION ?
STEPS INVOLVE IN TRANSCRIPTION.
RNA POLYMERASES.
HISTORY OF RNA POLYMERASES.
STRUCTURE OF RNA POLYMERASES.
SUB UNITS OF RNA POLYMERASES.
TYPES OF RNA POLYMERASES.
FUNCTION OF RNA POLYMERASES.
TRANSCRIPTION FACTORS INVOLVE IN EUKARYOTIC TRANSCRIPTION.
CONCLUSION.
REFERENCES.
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 ca.
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 ca.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
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.
Similar to Kathleen big Poster 2016 final copy (20)
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
Kathleen big Poster 2016 final copy
1. Examination of a Potential Open Reading Frame Co-transcribed with DksA in E. coli
Kathleen Barakat and Robert Osuna
Department of Biological Sciences, University at Albany, 1400 Washington Avenue, Albany, NY 12222
ABSTRACT
DksA is a critical transcription factor in Escherichia coli that plays an essential role in the response to
conditions of stress. DksA is an uncommon transcriptional regulator in that it binds directly to RNA
polymerase (RNAP) and not to DNA. Recent research has focused on acquiring detailed information on
how E. coli cells control the production of DksA in different growth conditions. There are three major
temporal promoters transcribing the dksA gene: P1, is highly expressed during early to mid exponential
growth phase, P2 is transiently expressed during entry into stationary phase, and P3 is a stationary phase-
specific promoter. The P3 promoter is dependent on the RpoS stress-dependent sigma factor. It is located
within the sfsA gene and is followed by what might possibly be a small open reading frame (ORF) of
unknown function. The goal of my work is to determine whether or not this ORF produces a protein
product that myight be co-expressed with dksA during stationary phase. To accomplish this, the segment
of DNA that transcribes the ORF was amplified by the polymerase chain reaction (PCR) such that a His6-
tag was added onto the C-terminal end of the putative ORF. The PCR product was purified, ligated on a
multicopy plasmid, and transformed into a wild-type E. coli strain. We then attempted to induce the
expression of the His6-tagged protein and purify it using Ni+-based affinity chromatography. Our results
repeatedly failed to detect induced levels of ORF polypeptide in different strains of E. coli. We therefore
reject the hypothesis that the putative open reading frame is translated into a polypeptide in vivo. These
results help clarify our picture of the roles played by the dksA promoters.
BACKGROUND
DksA is a transcription regulator that acts together with ppGpp
to mediate the Stringent Response
DksA Mediates its Effects By Binding to RNA Polymerase
CONCLUSION
RESULTS
Several temporal promoters transcribe dksA.
Hypothesis: The putative ORF encodes a polypeptide, which is co-expressed
with DksA during stationary phase
Matches to known Helix-Turn-Helix DNA binding motifs
MSVAAEGQRAVIFFAVLHSAITRFSPARHIDEKYAQLLSEAQQRGVEILAYKAEISAEGMALKKSLPVTL
I A S V A Q H V C L S P S R L S H L F R AraC
L Y D V A E Y A G V S Y Q T V S R V V N LacR
Q T K T A K D L G V Y Q S A I N K A I H Cro
T R K L A Q K L G V E Q P T L Y W H V K TetR
I K D V A R L A G V S V A T V S R V I N GalR
R A E I A Q R L G F R S P N A A E E H L LEXA
L L S E A Q Q R G V E I L A Y K A E I S ORF
Predicted ORF Polypeptide of 70 Amino Acids
Significance: Co-transcribed genes in bacteria are often involved in related
functions. As a putative polypeptide with a DNA-binding motif, the product of
ORF may be envisioned to collaborate with DksA in regulating gene expression
during stationary phase.
Approach:
To investigate whether ORF can be translated in vivo into a polypeptide, we
engineered a DNA construct with
• a C-terminal His6-tag on ORF
• an IPTG-inducible promoter (Ptac) transcribing ORF
Starvation/Stress
ppGpp
Starvation/Stress
ppGpp
Secondary Channel
Secondary Channel
DksA
DksA
Transcription Activation
Transcription Repression
DksA
ppGpp
ppGpp
Primer Extension Analysis
P2
P3
P1
S1-Nuclease Analysis
-154
-53
-475
1 2 3 4 50.5TGCA
Probe
Probe
Exp. Stat.
Hours of Growth
P1
P2
P3
A
B
C
1kbladder
CBA
Acrylamide gel electrophoresis
of PCR Products
PCR Strategy
*
*
100bpladder
Plasmids carrying Ptac-ORF
ControlPlasmid
5% Polyacrylamide Gel of candidate plasmids
carrying Ptac-ORF. Digestion with EcoRI and
HindIII releases a 340 bp DNA fragment containing
the Ptac-ORF-His6 construct.
Ni2+-IMAC Chromatography of Native Proteins
100 bp
200 bp
300 bp
400 bp
500 bp Ptac-ORF-His6
DNA insert
Plasmids carrying Ptac-ORF-His6 DNA
verified by restriction digestion
sfsA
sfsA
Grow Cells carrying:
-Plasmid with Ptac-ORF-His6
-Control Plasmid
Induce with IPTG
for 2 hours at 37°C
Collect and Lyse Cells
Bind to Ni2+-IMAC Affinity
Chromatography Column
Flow
Through
Wash Bound
Experimental Outline
Bound
SizeStandards
Lysate
Flowthrough
Wash
MW (kDa)
75
150
5
10
15
20
25
37
50
100
250
13 kDa
8.45 kDa expected size
Ni2+-IMAC Chromatography of Denatured Proteins
FlowThrough
PrecisionPlusLadder
ControlLysate
Ptac-ORF-His6Lysate
5
10
15
20
25
37
50
100
250
75
MW (kDa)
13 kDa
8.45 kDa expected size
Bound
SizeStandards
Control
Ptac-ORF
Ptac-ORF
Ptac-ORF
Control
Control
10 µg 20 µg 30 µg
5
10
15
20
25
37
50
100
250
75
MW (kDa)
8.45 kDa expected size
Proteins Expressed in ∆lon Strain
Proteins from ∆lon Strain Purifed by
Ni2+ - IMAC Chromatography
FT
FT
FT
FT
Wash
Wash
Wash
Wash
Bound
Bound
Bound
Bound
Ptac-ORF-His6Control
8.45 kDa
expected size
We see no evidence of Expression of a polypeptide of the expected size for OR(8.5 kDa)
after IPTG induction from a strong promoter and after several attempts to capture the
polypeptide using Ni2+ - IMAC affinity chromatography. Therefore, we reject the
hypothesis that ORF expresses a polypeptide in vivo. These results help clarify our picture
of the possible roles of the dksA promoters.