Summary Molecular Genetics & Biotechnology of Micro-organisms (G0F77A)
1 Lecture 1: Microbial biosensors and reporters
Biosensor = A device that uses a biological recognition element (like an
engineered microorganism) to detect a specific substance, producing
a measurable signal (a readout).
Reporter = a reporter refers to the system (Reporter gene) and the resulting
Reporter protein, which produces the final, measurable output signal
(the Readout) in response to a biological event.
1.1 Advantages
Whole-cell based biosensor
= biosensor where the
biological recognition and signal
production system is integrated
within a living cell (such as
bacteria). This uses the cell's
natural systems to process the
analyte signal and generate a
readout .
Comparison with other methods:
Microbial sensor Analytical/chemical Enzymes
methods
(Chip, HPLC/GC)
Low cost expensive expensive
Easy to use
Fast read out
Bioavailability
Bioaccessibility High selective
highly stable instable
High accuracy Hard purificationproces
1
,1.2 Design – integrating synthetic biology & microengineering
Almost the whole lecture explains
this figure in detail!!! Make sure
you always know where you are in
this figure. The subsections of the
lecture are added in this overview!
The sensing module senses the
ligand and needs to be very
selective and sensitive.
The signal processing module
can be added so only an output is
generated in certain scenarios like:
2 specific signals
1 signal needs to be present
and 1 signal has to be
absent
Etc.
The output module will create something we can detect like fluorescence,
luminescence, colorimetric of electrochemical.
1.2.1 Sensors/receptors (slide 19)
Bacterial sensing and signaling pathways are usually either one-component or
two-component systems. In a one-component system can the reporter gene
either be activated or is the reporter gene constantly inhibited and will the
inhibitor get repressed (derepression). A two-component system is usually over a
membrane and triggers a cascade inside the membrane.
2
,Bacteria can be engineered to sense and reported on other organisms diseases.
1) Thermal bioswitch
A temperature-dependent transcriptional
repressor (TSR) is a protein that is expressed
constantly but there function is dependent on the
temperature. At body temperature, the TSR will
inhibit the promotor of the GFP. When the temperature of the body rises
(fever), than will TSR stop inhibiting and GFP will be expressed.
Organism is healty Body temperature normal normal
Organism is sick body temperature is high (fever) GFP is expressed
2) Detection of inflammation
A probiotic E.coli gut bacteria is engineered to
detect thiosulphate (S2O32-). Thiosulphate will
bind the membrane of the bacteria, which will
trigger a cascade and express GFP. This is a
two-component system.
3) Pathogen detection by quorum sensing
When a pathogen has a certain population in an organism, it will secrete
chemical signals. These signals will interact with a modified E.coli which
will produce an antibiofilm factor, antimicrobial peptides or lysin to disrupt
the growth of the pathogen or biofilm.
1.2.2 Signal processing modules (slide 27)
We can add different mechanisms so
the output tells us more and more. So
can the output change in different
environments like light, heat, the
presence of ions,…
Examples:
1) Logic gates
= the building blocks of synthetic genetic circuits. They are engineered to
allow a specific biological output (expression of the reporter gene) only
under a defined combination of molecular inputs (signals).
Type of
Meaning
gate
AND There is an output signal if Input A AND Input B are both ON (or
present).
OR There is an output signal if Input A OR Input B OR both are ON
(or present).
XOR There is an output signal if Input A is ON OR Input B is ON, but
3
, NOT both ON (Exclusive OR)
NOR There is an output signal if Input A is OFF AND Input B is OFF
XNOR There is an output signal if Input A AND Input B are the same
(either both ON or both OFF)
NAND There is an output signal if Input A is OFF OR Input B is OFF
(meaning they are NOT both ON)
NOT There is an output signal if there is no input signal present (of
omgekeerd!)
2) Memory circuit
Memory circuits can convert a temporary signal into a longer-term cellular
response. After a while they are cheaper and have a low burden.
Example of a memory circuit cI protein and Cro protein
By default, the memory circuit is OFF and cI will bind the promotor (pR)
so transcription of Cro and LacZ isn’t possible. But when an
environmental trigger initiates the transcription of Cro, than will Cro
inhibit the transcription of cI. This will also start the transcription of lacZ
(β-galactosidase), which is our output signal. Even when the
environmental trigger disappears will the system not change back to the
default state and thus will the output signal stay. The memory circuit is
ON.
Recombinase -mediated
By default, the memory circuit is OFF. The expression of LacZ is not
possible because the sequence of specific parts of the gene is in
reverse. An environmental trigger will initiate the transcription of two
recombinases. These enzymes reverse the specific parts of the gene so
the LacZ is complete and can be expressed into β-galactosidase. The
memory circuit is ON because the DNA-change will stay forever even is
the environmental signal goes away.
4
1 Lecture 1: Microbial biosensors and reporters
Biosensor = A device that uses a biological recognition element (like an
engineered microorganism) to detect a specific substance, producing
a measurable signal (a readout).
Reporter = a reporter refers to the system (Reporter gene) and the resulting
Reporter protein, which produces the final, measurable output signal
(the Readout) in response to a biological event.
1.1 Advantages
Whole-cell based biosensor
= biosensor where the
biological recognition and signal
production system is integrated
within a living cell (such as
bacteria). This uses the cell's
natural systems to process the
analyte signal and generate a
readout .
Comparison with other methods:
Microbial sensor Analytical/chemical Enzymes
methods
(Chip, HPLC/GC)
Low cost expensive expensive
Easy to use
Fast read out
Bioavailability
Bioaccessibility High selective
highly stable instable
High accuracy Hard purificationproces
1
,1.2 Design – integrating synthetic biology & microengineering
Almost the whole lecture explains
this figure in detail!!! Make sure
you always know where you are in
this figure. The subsections of the
lecture are added in this overview!
The sensing module senses the
ligand and needs to be very
selective and sensitive.
The signal processing module
can be added so only an output is
generated in certain scenarios like:
2 specific signals
1 signal needs to be present
and 1 signal has to be
absent
Etc.
The output module will create something we can detect like fluorescence,
luminescence, colorimetric of electrochemical.
1.2.1 Sensors/receptors (slide 19)
Bacterial sensing and signaling pathways are usually either one-component or
two-component systems. In a one-component system can the reporter gene
either be activated or is the reporter gene constantly inhibited and will the
inhibitor get repressed (derepression). A two-component system is usually over a
membrane and triggers a cascade inside the membrane.
2
,Bacteria can be engineered to sense and reported on other organisms diseases.
1) Thermal bioswitch
A temperature-dependent transcriptional
repressor (TSR) is a protein that is expressed
constantly but there function is dependent on the
temperature. At body temperature, the TSR will
inhibit the promotor of the GFP. When the temperature of the body rises
(fever), than will TSR stop inhibiting and GFP will be expressed.
Organism is healty Body temperature normal normal
Organism is sick body temperature is high (fever) GFP is expressed
2) Detection of inflammation
A probiotic E.coli gut bacteria is engineered to
detect thiosulphate (S2O32-). Thiosulphate will
bind the membrane of the bacteria, which will
trigger a cascade and express GFP. This is a
two-component system.
3) Pathogen detection by quorum sensing
When a pathogen has a certain population in an organism, it will secrete
chemical signals. These signals will interact with a modified E.coli which
will produce an antibiofilm factor, antimicrobial peptides or lysin to disrupt
the growth of the pathogen or biofilm.
1.2.2 Signal processing modules (slide 27)
We can add different mechanisms so
the output tells us more and more. So
can the output change in different
environments like light, heat, the
presence of ions,…
Examples:
1) Logic gates
= the building blocks of synthetic genetic circuits. They are engineered to
allow a specific biological output (expression of the reporter gene) only
under a defined combination of molecular inputs (signals).
Type of
Meaning
gate
AND There is an output signal if Input A AND Input B are both ON (or
present).
OR There is an output signal if Input A OR Input B OR both are ON
(or present).
XOR There is an output signal if Input A is ON OR Input B is ON, but
3
, NOT both ON (Exclusive OR)
NOR There is an output signal if Input A is OFF AND Input B is OFF
XNOR There is an output signal if Input A AND Input B are the same
(either both ON or both OFF)
NAND There is an output signal if Input A is OFF OR Input B is OFF
(meaning they are NOT both ON)
NOT There is an output signal if there is no input signal present (of
omgekeerd!)
2) Memory circuit
Memory circuits can convert a temporary signal into a longer-term cellular
response. After a while they are cheaper and have a low burden.
Example of a memory circuit cI protein and Cro protein
By default, the memory circuit is OFF and cI will bind the promotor (pR)
so transcription of Cro and LacZ isn’t possible. But when an
environmental trigger initiates the transcription of Cro, than will Cro
inhibit the transcription of cI. This will also start the transcription of lacZ
(β-galactosidase), which is our output signal. Even when the
environmental trigger disappears will the system not change back to the
default state and thus will the output signal stay. The memory circuit is
ON.
Recombinase -mediated
By default, the memory circuit is OFF. The expression of LacZ is not
possible because the sequence of specific parts of the gene is in
reverse. An environmental trigger will initiate the transcription of two
recombinases. These enzymes reverse the specific parts of the gene so
the LacZ is complete and can be expressed into β-galactosidase. The
memory circuit is ON because the DNA-change will stay forever even is
the environmental signal goes away.
4
