Simultaneous determination of gene expression and enzymatic activity in individual bacterial cells in microdroplet compartments.
A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.
Shim
Jung-uk
JU
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, United Kingdom.
Olguin
Luis F
LF
Whyte
Graeme
G
Scott
Duncan
D
Babtie
Ann
A
Abell
Chris
C
Huck
Wilhelm T S
WT
Hollfelder
Florian
F
D000469:Alkaline Phosphatase / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D000097573:Red Fluorescent Protein
D000469:Alkaline Phosphatase; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein