Transcript PowerPoint - 2014 Science Interns

Polishing Your Prose
BNL, CCI, Summer ’13
Week 4
Mike Stegman
Metabolic Engineering of Alicyclobacillus
acidocaldarius to make Lactic Acid
Alicyclobacillus acidocaldarius is a gram-positive bacteria found in thermal features in
Yellowstone National Park. It grows at 60 °C and a pH of 4.0. A. acidocaldarius has been found
to aid in the production of lactic acid, a chemical that can be used to make biodegradable
plastic. Currently, the process for making lactic acid is costly and generates waste which
comes from the need to perform acid pretreatment on the plant matter that is used as a
starting material. After the acid pretreatment, the solution needs to be neutralized and
cooled before enzymes that break down sugars into lactic acid can be added. If A
acidocaldarius is used, the liquid from the acid pretreatment will not have to be cooled or
neutralized as extensively and the bacteria will break down the sugars directly into lactic acid,
cutting out intermediate steps while reducing waste and cost. In order for the bacteria to
make lactic acid, the cells must produce excess lactate dehydrogenase (LDH), an enzyme that
converts pyruvate into lactic acid during carbon metabolism. A. acidocaldarius does not make
excess LDH under normal conditions, so an LDH gene must be inserted. The purpose of this
study is to insert a plasmid containing an antibiotic resistance gene and a controllable LDH
gene into the bacteria to aid in the production of lactic acid. This is done by mating A.
acidocaldarius with Escherichia coli cells that contain the plasmid. The two types of bacteria
are grown overnight, combined, and then passed through a filter to get the cells in close
proximity. To select for cells with the plasmid, the filter is then incubated on agar at 37 °C
overnight, and the cells are suspended from the filter into liquid medium, plated on agar with
antibiotic added and incubated at 60 °C. This is done in hopes that the E. coli cells will
transfer the plasmid to A. acidocaldarius. Unfortunately, no plasmid transfer was seen. This
could be due to unfavorable conditions; mating at pH 5.5 and 37 °C, while optimal for E. coli,
is not optimal for A. acidocaldarius, making the cells incapable of receiving DNA. It is also
possible that the plasmid is transferred but can’t replicate in A. acidocaldarius. The goal of
expressing LDH in order produce lactic acid will not be met until a system for getting DNA into
the cell is developed. In the future, constructing a plasmid that contains native genes from A.
acidocaldarius may be helpful because it is likely that the plasmid will be more easily
accepted by the bacteria.
Metabolic Engineering of Alicyclobacillus
acidocaldarius to make Lactic Acid
Alicyclobacillus acidocaldarius, a gram-positive bacteria found in thermal features in Yellowstone
National Park at 60 °C and a pH of 4.0, aids in the production of lactic acid, a chemical that can be used to
make biodegradable plastic. Currently, the process for making lactic acid is costly and generates waste
which comes from the need to perform acid pretreatment on the plant matter that is used as a starting
material. After the acid pretreatment, the solution needs to be neutralized and cooled before enzymes
that break down sugars into lactic acid can be added. If A acidocaldarius is used, the liquid from the acid
pretreatment will not have to be cooled or neutralized as extensively and the bacteria will break down the
sugars directly into lactic acid, cutting out intermediate steps while reducing waste and cost. In order for
the bacteria to make lactic acid, the cells must produce excess lactate dehydrogenase (LDH), an enzyme
that converts pyruvate into lactic acid during carbon metabolism. A. acidocaldarius does not make excess
LDH under normal conditions, so an LDH gene must be inserted. This study sets out to insert a plasmid
containing an antibiotic resistance gene and a controllable LDH gene into the bacteria to aid in the
production of lactic acid by mating A. acidocaldarius with Escherichia coli cells that contain the plasmid.
The two types of bacteria are grown overnight, combined, and then passed through a filter to get the cells
in close proximity. To select for cells with the plasmid, the filter is then incubated on agar at 37 °C
overnight, and the cells are suspended from the filter into liquid medium, plated on agar with antibiotic
added and incubated at 60 °C. This is done in hopes that the E. coli cells will transfer the plasmid to A.
acidocaldarius. Unfortunately, no plasmid transfer was seen. This could be due to unfavorable conditions;
mating at pH 5.5 and 37 °C, while optimal for E. coli, is not optimal for A. acidocaldarius, making the cells
incapable of receiving DNA. It is also possible that the plasmid is transferred but can’t replicate in A.
acidocaldarius. The goal of expressing LDH in order produce lactic acid will not be met until a system for
getting DNA into the cell is developed. In the future, constructing a plasmid that contains native genes
from A. acidocaldarius may be helpful because it is likely that the plasmid will be more easily accepted by
the bacteria.
Advanced Protein Purification and Crystallization Identification
Using High Performance Chromatography
The purpose of the research performed by protein purifiers in the Biosciences
Division at the Structural Biology Center (SBC) is to collect a sample of protein
completely free of contaminates so that a correct crystallized form of that protein
can be obtained. By determining a protein’s three dimensional structure,
researchers are able to understand its functionality within its natural state and the
affects/consequences it has based on its tertiary folding. The SBC has established
three different teams working cohesively, whose sole purpose is to develop
proteins, purify protein and to determine the validity of that protein’s structure.
Using synchrotron X-ray crystallography, the three dimensional structure of protein
can be determined. Based on the SBC procedure and protocol, all target proteins
must fall within a pre-determined range of solubility and expression to ensure the
best crystal formation. Since only the purest proteins produce the strongest
crystals, the SBC requires the use of high performance chromatography
workstations AKTA Xpress and IMAC 2 to ensure as little contamination as possible.
This procedure should ensure that once the purified target proteins are exposed to
certain conditions it should solidify into a crystallized structure, if not, additional
protocols will be implemented to tease forward solidified structures. Once a
crystal is formed and identified it is passed on to the crystallographers for
structure analysis, the third stage of the SBC protocols.
Advanced Protein Purification and Crystallization Identification
Using High Performance Chromatography
The Biosciences Division of the Structural Biology Center (SBC) has established
three teams solely to develop a protein, to purify the protein, and to determine
the validity of that protein’s structure by collecting a protein sample completely
free of contaminates so that a correct crystallized form of that protein can be
obtained. By determining a protein’s three dimensional structure, researchers are
able to understand its functionality within its natural state and the
affects/consequences it has based on its tertiary folding. Using synchrotron X-ray
crystallography, the three dimensional structure of protein can be determined.
Based on the SBC procedure and protocol, all target proteins must fall within a predetermined range of solubility and expression to ensure the best crystal formation.
Since only the purest proteins produce the strongest crystals, the SBC requires the
use of high performance chromatography workstations AKTA Xpress and IMAC 2 to
ensure as little contamination as possible. This procedure should ensure that once
the purified target proteins are exposed to certain conditions it should solidify into
a crystallized structure, if not, additional protocols will be implemented to tease
forward solidified structures. Once a crystal is formed and identified it is passed on
to the crystallographers for structure analysis, the third stage of the SBC protocols.
Detecting Trace Ambient Pesticides in Real Time Using
Single Particle Aerosol Mass Spectrometry (SPAMS)
Pesticides, by nature, are toxic substances, and may cause unintentional harm if improperly
controlled. Given that pesticides are fairly ubiquitous, with wide use in agriculture and the
household, and considering the potential for harm that pesticides pose to non-target
organisms such as pets and humans, the detection of pesticides using rapid and effective
means is particularly attractive. In this study, Single Particle Aerosol Mass Spectrometry
(SPAMS) was used to demonstrate the rapid detection of five commonly used pesticides,
often used in agricultural and home-and-garden applications. These include permethrin
(pyrethroid class), malathion and dichlorvos (organophosphate class), imidacloprid
(chloronicotinyl class) and carbaryl (carbamate class). Analytical standards of each compound
were used to prepare a liquid solution through mixture with water or ethanol. The final liquid
sample solution was aerosolized in a collision nebulizer to create particles for analysis in the
SPAMS instrument. The resultant dual-polarity time-of-flight mass spectra were then
analyzed to identify the characteristic peaks of the compound in the sample. In addition,
samples of common household items containing pesticides, namely Raid™ Ant Killer 16 spray,
containing permethrin, and a canine flea collar, containing carbaryl, were analyzed in their
original state using SPAMS, without any further sample preparation. The characteristic peaks
of the active pesticides in these samples were identified using the mass spectra obtained
earlier from the pesticide analytical standards. By successfully identifying pesticides in
analytical standards and in household items, it is demonstrated here that the SPAMS system
is capable of pesticide detection in numerous agricultural and environmental situations.
Commas
Only four comma rules to keep in mind (and the last
two are related). Use a comma:
• Before and, but, for, or, nor, yet, still, when joining
independent clauses.
• Between all terms in a series, including the last two.
• To set off parenthetical openers and afterthoughts.
• Before and after parenthetical insertions (use a pair
of commas).
From The Practical Stylist, Sheridan Baker.
Before and, but, for, or, nor, yet, still,
when joining independent clauses.
•
•
•
•
Think of ,and and ,but as a unit, perfectly equivalent to the . and the ; as a buffer between independent
clauses.
.
He was tired. He went home.
;
He was tired; he went home.
, and He was tired, and he went home.
•
•
He hunted the hills and . . . .
He hunted the hills, and . . . .
•
•
Wear your jacket or coat.
Wear your jacket, or you will catch cold.
•
•
It was strong yet sweet.
It was strong, yet it was not unpleasant.
• Next morning when the first light came into
the sky and the sparrows stirred in the trees,
when the cows rattled their chains and the
rooster crowed and the early automobiles
went whispering along the road, Wilbur
awoke . . . .
from Charlotte’s Web, E. B. White
Between all terms in a series, including
the last two.
• words, phrases, or clauses in a series
• to hunt, to fish, and to hike
• He went home, he went upstairs, and he could
remember nothing.
• He lost all his holdings, houses and lands.
• I met two scientists, Jill and Jack.
• I met two scientists, Jill, and Jack.
To set off parenthetical openers and
afterthoughts.
•
•
•
•
•
However, he liked it.
Inside, everything was chaos.
For several reasons, he stayed home.
Although he never looked bored, he kept on talking.
If it is not too much trouble, punctuate accurately.
• However he tried, he could not do it.
• However, he tried.
• However he tried, he could not do it; however, he tried.
• He stayed home for several reasons.
• For several reasons he stayed home.
• Punctuate accurately if you can.
Before and after parenthetical
insertions (use a pair of commas).
• The car, an ancient Packard, is still running.
• April 10, 1980, is fine.
• John, my friend, will do what he can.
• The taxes, which are reasonable, will be paid.
• The taxes which are reasonable will be paid.
Stopping by Woods on a Snowy Evening
Robert Frost