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Marker
Gene Monthly Newsletter
August, 2007
Volume
7, Number 8
©
Copyright MGT, Inc., 2007. Published by
Marker Gene Technologies, Inc., The University of Oregon Riverfront Research
Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA. All rights reserved. For information on the use or copying of the
material contained in this document, please contact us at techservice@markergene.com. Please see below for subscription
information and updates. This
newsletter is labeled as an ADVERTISEMENT in accordance with the
CAN-SPAM act of 2003, S.877 Public Law: 108-187.
Whole Genome Transplant –
Toward a Synthetic Life Form.
Craig Venter, and colleagues at the J. Craig Venter
Institute and Synthetic Genomics, Inc. in Rockville, Maryland, have published a
proof-of-concept experiment in which they were able to completely transplant
the whole bacterial genome from one species into a different species, so that
the recipient organism became phenotypically and genotypically identical to the
original donor organism. In this
particular experiment, Mycoplasma mycoides Large Colony (LC) served as
the donor of the DNA, and Mycoplasma capricolum was the recipient. Since manipulation of whole chromosomes in
solution can expose the DNA to shear forces and other problems, the DNA was
suspended in agarose blocks during manipulations. The proteins were first removed by enzyme and detergent treatment
from the M. mycoides LC DNA, resulting in “naked” DNA. Then this intact DNA was incubated with M.
capricolum cells, that had been subjected to low pH culture,
centrifugation, and nucleotide starvation.
A polyethylene glycol transformation technique was used to transfer the
whole genome DNA in b-agarase-treated
plugs into the M. capricolum cell “shells”. As the cells fused, they sometimes encapsulated the donor DNA
chromosomes.
Since the original M. mycoides LC cells contained both cloned tetR
and lacZ marker genes, treating the cells with the antibiotic
tetracycline was used to select for new transformants. The new cells were also stained for lacZ
activity using X-Gal to develop characteristic blue colonies, and these methods
showed that about 1:150K had obtained the correct phenotype. They also performed 2D-SDS PAGE proteomic
analysis tests to confirm the identity of the new organism. These results demonstrate that the
techniques are available that can be used for whole genome transplantations. This process is a prelude to what the researchers
hope will be a new area they call “synthetic genomics” in which synthetic DNA
that code for completely new genomes will be used to construct entirely new
microorganisms. These may enable the production of synthetic microbes for
potential green energy sources, pharmaceuticals, chemicals or even
textiles.
·
Lartigue C, Glass JI,
Alperovich N, Pieper R, Parmar PP., Hutchison CA., Smith HO, and Venter JC,
(2007) “Genome Transplantation in Bacteria: Changing One Species to Another.”
Science 317: 632-637.
·
Glass JI, Assad-Garcia
N, Alperovich N, Yooseph S, Lewis
MR, Maruf M, Hutchison CA, Smith HO,
Venter, JC, (2006) “Essential genes of a minimal bacterium.” Proc. Natl.
Acad. Sci. USA 103(2): 425-30.
·
Smith HO, Hutchison CA,
Pfannkoch C, Venter, JC, (2003) “Generating a synthetic genome by whole genome
assembly: phiX174 bacteriophage from synthetic oligonucleotides.”
Proc.Natl.Acad.Sci.USA 100(26): 15440-15445.
· http://www.syntheticgenomics.com/ ; http://www.jcvi.org/
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Bacterial Luciferase Assay in Mammalian Cells. The
lux operon, encoding bacterial luciferase, is unique in that it also
encodes genes for generating its own substrate, preferentially a long-chain
aldehyde, such as decanal. As such,
the lux system has advantages over other biolumininescent systems like
firefly luciferase, since stable transformation can result in self-contained
light emission without the requirement for exogenous substrates or
external activation over long periods or for many cell lineages. A reporter system encoding the entire luxCDABE operon would allow such
autonomous measurement of expression, eliminating the need for cell lysis or
substrate addition. LuxA and luxB
encode the two subunits of the dimeric luciferase protein. Recently, researchers at the University of
Tennessee, Knoxville, have produced codon-optimized sequences of the luxA
and luxB genes from the bacterium Photorhabdus luminsecens to
facilitate higher expression of bacterial luciferase in mammalian cells. Cell extracts of HEK293 stable transformants carrying
one optimized subunit sequence and one wild-type subunit sequence saw a
6-fold increase in luminescence over stable transformants carrying the
wild-type sequence for both subunits, upon addition of the substrate decanal
and cofactor FMNH2. Stable
transformants carrying optimized sequences of both subunits resulted in even
greater bioluminescence. Work is
underway to express the entire lux operon for use in providing continuous
bioluminescent expression. We are
currently developing mammalian expression vectors that encode both the luxA
and luxB subunits of the bacterium Vibrio harveyi as well as other operon elements for
versatile use in many mammalian cell systems. For more information regarding these vectors and assays of
bacterial luciferase, please visit our website
or see the references below. ·
Patterson SS, Dionisi
HM, Gupta RK, Sayler GS, (2005) “Codon optimization of bacterial
luciferase (lux) for expression in mammalian cells.” J. Ind. Microbiol. Biotechnol., 32: 115-123. ·
Cohn DH, Mileham AJ,
Simon MI, Nealson KH. (1985) “Nucleotide Sequence of the luxA
Gene of Vibrio harveyi and the Complete Amino Acid Sequence of the
α Subunit of Bacterial Luciferase.”
J. Biol. Chem. 260(10):
6139-6146.
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Live Cell FDG Plate Assay
for Mammalian Cells. The ability to
monitor lacZ activity in live cells grown in culture is of interest
for transformation studies, studies on the effect of secondary gene
expression or regulation by other cis- or trans-acting factors. Fluorescein
di-b-D-Galactopyranoside (FDG, M0250)
is probably the most sensitive fluorogenic reagent available for use in
measuring lacZ activity in either live cells, by FACS analysis or in
cell lysates. We supply the reagents
and protocol in a kit form with other cofactors and inhibitors useful for
direct cellular analysis in our MarkerGeneTM
in vivo lacZ ß-Galactosidase Intracellular Detection Kit (Product
M0259). Since this reagent and protocol are non-toxic, the stained cells can be
recovered for later analysis or expansion as desired. Appropriate sterile conditions should be
maintained if later expansion of the cells is required. In order to directly measure the expression level of the lacZ marker gene in cells grown in culture, cells (adherent or non-adherent) are cultured in multi-well tissue culture plates (such as BD Falcon clear, flat bottom plates; with 6, 12, or 96-wells). It is recommended that a few plate wells be left empty that contain no cells as blank control wells. Ideally, a sufficient number of wells should be available so that assays can be performed in triplicate using approximately 1 x 106 cells per well (12-well plates). Prior to performing assay, all media should be replaced with serum-free growth medium (such as DMEM or RPMI 1640) without antibiotics. The presence of serum or antibiotics in the medium may affect results. Cells may be incubated in the serum-free medium for several hours (up to 24 hours) prior to analysis of ß-galactosidase. The Fluorescent Substrate Reagent (Part 0259-001) is added to wells so that final concentration in the medium is 500μM (1/20 volume of medium in well). Reference Standard (Part 0259-002) may also be added to additional wells at varying concentrations, for use in optimizing reading conditions or creating a calibration curve. Record fluorescence (EX/EM: 490/514) using a microtiter plate reader with appropriate excitation and emission filter sets. Fluorescence may either be recorded continuously, or at defined intervals, ranging from several minutes to several hours. When measurements are not being taken, cells should be placed in an incubator (37oC) under appropriate CO2 and humidification. Representative results of such an assay are shown below for NIH3T3 and CREBAG2 (lacZ+) cells. Please see our website or contact our technical assistance staff for more information about these assay methods.
Adherent mouse fibroblast
tumor cell lines, CRE BAG 2 (lacZ stable transformants), and NIH 3T3 (lacZ negative) were cultured to 50% confluency in 12-well
tissue culture plates (BD Falcon clear, flat bottom). Media was replaced with Dulbecco’s
Modified Eagles Medium (DMEM) containing no serum or antibiotics (1mL). Plate wells were also prepared containing
medium only (no cells). Cells were
incubated (37oC, 5% CO2) for 24 hours. Fluorescent Substrate Reagent (Part
0259-001) was added to all wells to a final concentration of 500μM
(50μL). Fluorescence was
recorded using a Perkin Elmer HTS7000 Plus Bio Assay Reader, using 485nm
excitation and 535nm emission filters.
Readings were taken at 1, 3, and 5 hour intervals. Assays were performed in triplicate and
averaged, error bars represent standard error. |
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In normal serum the concentration of lipase is quite low. But in acute pancreatitis and in pancreatic carcinoma a rise in serum lipase activity occurs, with a mean increase being about 50 times that of normal values. Such a rise in the serum lipase content is also found in acute and chronic renal diseases. In general, because of the low levels, most people performing lipase assays in serum use samples derived from these conditions, or measure lipase activity with serum that contains added lipase. But sensitive measurement of lipase activity in serum can be accomplished using the fluorescent substrate 1,2-Dioleoyl-3-(pyren-1-yl)decanoyl-rac Glycerol (M0258). Upon enzymatic cleavage, the fluorescent fatty acid, pyrenedecanoic acid (M0274) is released. The method, however, is slightly different than that described in our live cell or cell-lysate methods (i.e. the M0612: MarkerGeneTM Fluorescent Lipase Assay Kit) and involves an added extraction step to quantitate the released fatty acids according to the procedure of Salvayre (Salvayre, et al., 1986). Basically this method involves mixing 90 microliters of the substrate (prepared as an emulsion in a mixture of triolein and colipase [0.3 mM triolein, 26 mmol of Tris buffer (pH 9.0), and 3 mg of colipase per liter with 5 x 15 sec. ultrasonications] with 10 uL of serum (pure or diluted in water) and incubation at 30oC for 10 min. The reaction is stopped by adding an extraction solvent system of 1.5 mL of chloroform/methanol/heptane (125/140/100 by vol) and 0.5 mL of 0.5 M carbonate buffer, pH 10.5. After mixing and centrifuging, the liberated fatty acid extracted into the upper aqueous phase is measured fluorometrically by HPLC or TLC or by spectrofluorometer (excitation 342 nm, emission 398 nm) calibrated by using a standard of pyrenedecanoic fatty acid prepared exactly as in the assay conditions. Lipase activities are calculated as: Lipase, U/L = F/Fs x (ED/t) x 10; where F = fluorescence of the pyrenedecanoic acid liberated in the assay (corrected by subtraction of the blank), Fs = fluorescence of 1 pmol of pyrenedecanoic acid as measured under identical assay conditions, ED = enzyme dilution and t = time of incubation, in min. Activity (U) = mmol of total fatty acids liberated per minute and per liter of serum. Activity (mULL) = nmol of pyrenedecanoic acid liberated per minute per liter of serum. For more information about these techniques, please see our website or see the references below.
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