1. Plasmids containing fragments of the yeast prion protein Sup35 and GFP were mutated using site-directed mutagenesis to incorporate the unnatural amino acid p-benzoyl-L-phenylalanine (pBpa) at specific tyrosine sites.
2. The plasmids were transformed into E. coli cells containing the machinery for pBpa incorporation, and protein expression was induced to produce Sup35-GFP fusions containing pBpa.
3. SDS-PAGE and fluorescence microscopy confirmed the expression of full-length and truncated fusion proteins, and that GFP fluorescence was maintained after mutagenesis, suggesting pBpa incorporation did not greatly perturb protein structure.
Introduction.
History.
Central dogma.
Mechanism of protein synthesis.
Transcription.
Process of transcription
translation
Step of translation
Activation of amino acid.
Transfer of amino acid to tRNA.
Initiation of polypeptide chain
Elongation of polypeptide chain
Translocation
Termination of polypeptide chain
processing of released polypeptide chain
Main difference between protein synthesis in prokaryotes and eukryotes
Conclusion
Reference
Introduction.
History.
Central dogma.
Mechanism of protein synthesis.
Transcription.
Process of transcription
translation
Step of translation
Activation of amino acid.
Transfer of amino acid to tRNA.
Initiation of polypeptide chain
Elongation of polypeptide chain
Translocation
Termination of polypeptide chain
processing of released polypeptide chain
Main difference between protein synthesis in prokaryotes and eukryotes
Conclusion
Reference
Assessing post induction cellular response in a recombinant e. coli lactose i...Clark Hartsock
Assessing post-induction cellular response in a recombinant E. coli lactose inducible system by monitoring β-galactosidase levels; A paper written by me and Jeff Lewis
Sugars are molecules of fundamental importance for life on earth. Sugars act as primary carriers of captured energy from the sun. Sugars not only fuel cellular carbon and energy metabolism but also pay pivotal role as signaling molecules and sugar status modulates & coordinates internal regulators that govern growth and development. The genes involved in production of carbon from photosynthesis with its utilization, mobilization and allocation in various tissues at different developmental stages are highly regulated by sugars. In most plants, sucrose (Suc) is the end product of photosynthesis for translocation from the source to heterotrophic sinks through the sieve element/companion cell complex of the phloem.
In this presentation we will look the different pathways that initiates and propagates a serial cascade events for prpper cellular response, i hope to be intrested, and please if there any suggest do not hasitate for writting a replay to me, thanks
Assessing post induction cellular response in a recombinant e. coli lactose i...Clark Hartsock
Assessing post-induction cellular response in a recombinant E. coli lactose inducible system by monitoring β-galactosidase levels; A paper written by me and Jeff Lewis
Sugars are molecules of fundamental importance for life on earth. Sugars act as primary carriers of captured energy from the sun. Sugars not only fuel cellular carbon and energy metabolism but also pay pivotal role as signaling molecules and sugar status modulates & coordinates internal regulators that govern growth and development. The genes involved in production of carbon from photosynthesis with its utilization, mobilization and allocation in various tissues at different developmental stages are highly regulated by sugars. In most plants, sucrose (Suc) is the end product of photosynthesis for translocation from the source to heterotrophic sinks through the sieve element/companion cell complex of the phloem.
In this presentation we will look the different pathways that initiates and propagates a serial cascade events for prpper cellular response, i hope to be intrested, and please if there any suggest do not hasitate for writting a replay to me, thanks
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.
1.Receptors Link to other Enzymatic Activity.
2.Pathway of Intracellular Signal Transduction.
3.The Cyclic AMP pathway4.Cyclic GMP pathway
5.Phospholipids and Ca2+
6.The PI3-Kinase /Akt and mTOR pathways.
7.MAP Kinase Pathway.
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.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.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.
I am Mark T. I am a Molecular Biology Assignment Expert at nursingassignmenthelp.com. I hold a Masters’ in Medical Biotechnology, from Arizona State University, USA. I have been helping students with their assignments for the past 8 years. I solve assignments related to Molecular Biology.
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Similar to Tomasz Pawlak, TIGP SI poster, 2015 (20)
1. Methodology: Plasmid preparation
• Primers for the double stop codon TGATAA, omega GFP, Y13 and Y49 mutants, for pSup35(1-40) and (1-80)
were designed, based on the genetic code of Escherichia coli, with the addition of GFP, Sup35 (yeast),
Strep 2 and 6xHistidine tag.
• Plasmids were mutated through site-directed mutagenesis (using QuickChange kit protocol).
• Plasmid lengths checked via 1% agarose gel.
• Competent Escherichia coli transformed with the obtained plasmid. Transformed cells incubated overnight at
37oC.
• Two colonies inoculated into 3ml LB broth (+1X Ampicillin). These were incubated the following night.
Plasmids purified and mutations confirmed via DNA sequencing.
• Subcloning with the original plasmid was done to make sure no unwanted mutations took place.
• Restriction digestion, followed by DNA sequencing confirmed our mutations and the integrity of the rest of the
plasmid.
• From there, we repeat mutagenesis, this time for GFPΩ, onto TGATAA-mutated plasmid (40 only, as 80
mutagenesis failed). Result was confirmed in a similar manner (plasmid purification, digestion, sequencing).
Mutagenesis for GFPΩ on TGATAA was done because later on, we could not cut out GFPΩ, without the
double stop codon.
• Mutagenesis was repeated for Y13 in (1-40), Y13 and Y49 in (1-80).
• As GFPΩ(80) failed, we used subcloning to insert GFPΩ(40) into Sup35(80)
• We combine TGATAA with Y13(40), Y13(40) and Y(49) through subcloning.
• Competent cells were transformed with the mutations and results were confirmed with restriction-digestion
and sequencing.
- Each of the plasmids (Ampicillin resistant) were transformed into OverExpress C41(DE3) competent
cells containing pSupBpaRS-6TRN (Chloramphenicol/Cam resistant). Cells were then plated on LB
plates supplemented with Cam and Amp, and incubated at 37°C overnight.
Overnight colonies carried a tRNABpaCUA and amino-acyl tRNA synthetase specific to pBpa
(BpaRS). The BpaRS will aminoacylate the tRNABpaCUA, the anticodon of which is complementary
to amber condon.
Protein expression
Three colonies from each plate were inoculated into LB/Amp/Cam/pBpa broth, LB/Amp/pBpa (-Cam)
broth and LB/Amp/Cam (-pBpa) broth respectively. The latter two acted as controls.
After incubation at 37°C, 120 µl of overnight culture were subcultured, until OD600=0.6-0.8. A final
concentration of 1mM IPTG was added to the media, to induce expression of BpaRS/tRNA pair and
Sup35-GFP-StrepII-His mutants. Induction and incubation (37oC) took place for 3h. The cell-lysate
proteins were visualized by InstantBlue Dye via SDS-PAGE.
Results and discussion:
• Post induction, GFP mutants were viewed under the microscope. Following images were taken,
showing that GFP functionality (Figure4 & 5. a) – CAM, b) – pBpa, c) Fully induced):
• SDS-PAGE results of mutated cells show expected bands, plus background proteins. For comparison
to the original induced Sup35, refer to Figure 5
aReference:
1. Addition of a photocrosslinking amino acid to the genetic code of Echerichia coli, J. W. Chin, A. B.
Martin, D. S. King, L. Wang, P. G. Schultz; 2002, Proceedings of the National Academy of
Sciences of the United States of America.
2. Benzophenone Photophores in Biochemistry, G. Dormán, G.D. Prestwich; 1994, Biochemistry
3. Prion, Wikipedia, https://en.wikipedia.org/wiki/Prion
4. The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination, J.
Xie, L. Wang, N. Wu, A. Brock, G. Spraggon, P. G. Schultz; 2004, Nature Biotechnology
In vivo incorporation of para-benzoyl-l-phenylalanine,
an artificial amino acid, into protein Sup35, a yeast prion
Tomasz Pawlak, Chan Wai Shan and Chih-Yen King
1. Tyrosine codons
TAC, to be mutated
Figure 2: Original inserts, contain yeast Sup35 fragments, GFP, Strep II (purification), His tag
(purification), Amber/Ochre - original stop codons
Introduction:
Prions are proteins with multiple structurally unique
conformations, at least one of which can recruit
other, correctly folded prion proteins, resulting in the
formation of protein aggregates. These misfolded
proteins form amyloid fibrils. Formation and
propagation of these fibrils is closely associated with
several pathologies, such as Alzheimer’s Disease,
Kuru, Huntington’s Disease and Parkinson’s Disease
in humans and “Mad Cow’s” Disease (Bovine
Spongiform Encephalopathy) and Scrapie in other
mammals – cows and sheep respectively. These
protein-like infectious particles have fungal
equivalents, which can be used to investigate and
model folding patterns and progression of amyloids in
general. Sup35 is a translation termination factor in
S. cerevisiae which forms a complex with other
release factors.
Sup35 recognizes a stop codon, initiating polypeptide
release.
When infected with [PSI+], a self propagating
misfolded variant of Sup35 (201 amino acids), the
volume of functional Sup35p decreases, potentially
allowing translation to continue. This can result in a
non-functional, nonsense protein. Depending on the
genetic background, [PSI+] yeast can fare better
than non-infected yeast. [PSI+] yeast, still capable of
reproducing, infects its daughter cells. In a sharp
contrast to mammalian prion diseases, yeast prions
can be a positive influence on the cell. In this project,
Sup35 fragments (1-40) and (1-80), which are
capable of infecting “healthy” proteins were mutated,
as to allow an unusual amino acid, pBnp (para-
benzoyl-l-phenylalanine) to be incorporated into the
segments
Incorporation of unnatural amino acids, such as
photo linkers or heavy ions is a useful tool in
investigating protein interactions. Para-Bpa is capable
of cross-linking one Sup35 fragment to another,
proximal and sterically accessible peptide backbone,
under UV and was chosen due to the high fidelity of
that reaction. In (1-40) and (1-80), Tyr13 and Tyr13 &
49 respectfully, were changed to amber codons (UAG
instead of UAC, in both cases), to facilitate a
tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair,
improted from Methanococcus jannaschii, capable of
inerting pBpa into the polypeptide. Omega tyrosine
residue was mutated in the same manner in the
Green Fluorescent Protein, which was attached to
the construct as a control. If mutated GFPΩ
maintains its function, we can assume that this
mutation does not greatly disturb Sup35.
2. Primers with TAG
mutation are inserted
3. Polymerase Chain
Reaction (PCR)
5. Incorporation of the single strand
mutation into E. coli, plus ligation
4. Digestion of the
original plasmid
Scheme 1: Site-directed mutagenesis
Figure 1: (a)BpaRS aminoacylating
tRNABpa
CUA and (b)tRNA incorporating
pBpa during translation
pBpa
A U C
U A G
messenger RNA
Ribosome
growing polypeptide a)
b)
tRNA (M. jannaschii)
OR +( )
Scheme 3: Plasmid selection for protein expression
1 2 3 4
IPTG X √ √ √
pBpa √ √ X √
Cam (0.5x) √ √ √ X
a) Sup35(40) GFPΩ,
-CAM
b) Sup35(40) GFPΩ
-pBpa
c) Sup35(40) GFPΩ,
induced
a) Sup35(80) GFPΩ,
-CAM
b) Sup35(80) GFPΩ
-pBpa
c) Sup35(80) GFPΩ,
induced
Figure 4: Sup35(1-40) GFPΩ functionality Figure 5: Sup35(1-80) GFPΩ functionality
• From our results: lane 1 shows uninduced cells,
on all gels. No Sup35 band can be seen.
• Lane 2: induced Sup35-containing cell lysate,
shows bands at expected positions (1-40:
~33kDa, 1-80 ~38 kDa). Full length Sup35
fragments are annotated with a red line.
-In all but 1 case(Sup35(40) GFP), a band
can be seen corresponding to lane 4, i.e.
“truncated” polypeptide. Sup35 (80) should show
a similar pattern to (40), as the “truncated” –
early termination – line should be visible. We
cannot tell why that is not the case. This band
should only be seen in GFP mutants, as Sup35
mutants forming truncated peptides are too
short to be visualized here (<10 kDa).
• Lane 3, E. coli grown without pBpa. They show no
Sup35 presence, similarly to uninduced samples,
except for slightly lower bands. Additionally, GFPΩ
show truncated GFP bands. Due to the lack of pBpa,
translation ends, producing ~30kDa (40) and ~35kDa
(80) truncated peptides. Seen next to the green line.
• Lane 4, without Cam, selective pressure is gone and
Amp resistant bacteria grow preferentially. This
provides a negative control for GFPΩ.
Summary
• We’ve incorporated pBpa into Sup35(1-40) and (1-80).
• We’ve shown that pBpa can be incorporated into another protein –
GFP, while maintaining GFP’s function.
• Sup35 can be mutated at multiple positions.
• We’ve shown that pBpa doesn’t have a great impact on GFP
stability. Assuming that a peripheral tyrosine region of Sup35 is
mutated, it should not disturb protein’s conformation.
• We could use the same mutants for other artificial amino acids, eg.
p-iodo-l-phenylalanine, which could be visualized through Cryo EM.
• If we were to continue with pBpa, we could use UV and mass
spectrometry to detect where exactly the residues are, in relation
to other amino acids.
~180 kDa
~130
~70
~55
~40
~35
~25
~15
~10
Induced Sup35(1-40)
Induced Sup35(1-80)
~100
Figure 6: Original Sup35, induced not purified
1 2 3 4
Sup35(80) Y49
1 2 3 4
Sup35(80) GFPΩ
1 2 3 4
Mutation : Sup35(40) GFPΩ
1 2 3 4
Sup35(40) Y13
1 2 3 4
Sup35(80) Y13
Figure 7: SDS-PAGE of mutated Sup35 strands
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
~10
~15
~25
~35
~40
~55
~70
100 kDa
~10
~15
~25
~35
~40
~55
~70
~100 kDa
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
Scheme 2: Site-directed mutagenesis
Figure 3: pBpa structure