Next we need to ligate the R-vector with the E-insert
with DNA Ligase
Ligation of R and E BioBricks
XbaI
SpeI
ACTAGT
TGATCA
A
TGATC
ACTAGA
TGATCT
CTAGA
T
PstI
PstI
CTGCAG
GACGTC
TCTAGA
AGATCT
TGCAG
C
CTGCAG
GACGTC
C
GACGT
CTGCAG
GACGTC
Measuring DNA concentration for ligation reaction
We want 10 ng of vector mass. How much insert mass do we need?
25.4 ng E-insert = 6 X (898 / 2120) X 10 ng R-vector
10 ng R-vector / 7.5 ng/ul = 1.33 ul
25.4 ng E-insert / 18.4 ng/ul = 1.38 ul
Expression strain
That expresses lacI
Successful Transformants
Relative GFP Fluorescence of E. coli
induced with IPTG vs. uninduced
Overnight culture of E. coli induced with IPTG
Josh’s mid-log culture of E. coli induced with IPTG
Measuring fluorescence in 96-well plates
with a Victor3 plate reader
96-well plate
96-well plate assay example
Rate of change of GFP concentration over time
Entus et al. 2007. Systems and Synthetic Biology 1: 119-28
Fluorescence (cell density normalized) vs. IPTG concentration
Entus et al. 2007. Systems and Synthetic Biology 1: 119-28
Measuring gene expression at the single cell level
Rosenfeld et al., 2005. Science 307: 1962-5
Stochastic gene expression in single cells
¾ Studies on populations mask behavior of individual cells (often variation is present
in genetically identical cell populations exposed to the same environment)
¾ Gene expression “noise” is thought to contribute to this phenotypic variation
• Extrinisic noise = fluctuations in cellular components (global effect)
• Intrinisic noise = stochastic nature of gene expression process (local effect)
¾ Single-cell studies are important for understanding how stochastic gene expression
affects behavior of genetic circuits and to design noise-tolerant circuits
Example: noise propagation in gene networks
Pedraza JM and van Oudenaarden A, 2005. Science 307: 1965-9.
Methods for measuring single-cell gene expression
Fluorescence time-lapse microscopy
(e.g. agar-pad imaging, low throughput)
Measures and tracks gene
expression dynamics of individual
cells on a glass slide or dish, but only
small numbers of cells can be
observed
Rosenfeld et al., 2005. Science 307: 1962-5
Flow cytometry
(high throughput)
Simultaneously measures several
parameters of up to 10,000 cells
per second with high precision, but
only at a single timepoint
http://www.lanl.gov/news/1663/images/bird4lg.jpg
Microfluidic devices
Combines best features of fluorescence time-lapse microscopy
and flow cytometry together
¾Tracks gene expression changes in individual cells over several generations
(greatly extends time-lapse fluorescence microscopy experiments)
¾ Measures several parameters of thousands of cells per second
¾ Allows for precise control of microenvironment (environmental inputs)
Longo and Hasty, 2006. Mol. Sys. Bio. 1: 1-6