Synthetic Biology Notes

General Terms

• Ex vitro

In vivo (“within the living)– experimentation using a whole, living organism (e.g. animal testing, clinical trials) ex vivo (“out of the living”) –

experimentation/measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions

In vitro (“within the glass”) – experimentation using a controlled environment (ex. Test tube/petri dish)

in silico– performed on computer or via computer simulation

A SYNTHETIC GENETIC EDGE DETECTION PROGRAM

Tabor 2009

System• Mask filters light detected by bacteria• bacteria/ lawn detects edges + gives visual output

Concept of Detection System• Bacteria in dark produce signal (green circle)• Signal diffuses• Only bacteria in light respond to signal;

• Response = expression of visible reporter gene

NOT light box

cell-cell communication

inverter

X AND (NOT Y)

• light, no signal, no pigment• light, signal, pigment• No light, no signal, no pigment• no light, signal, no pigment

IF NOT lightproduce signal;

IF signal AND NOT (NOT light)produce pigment;

Y = 0 when there’s light.

light sensitive protein

cph8 = [Cph1]+[EnvZ]• Cph1 = photoreceptor domain of Synechocystis• EnvZ = kinase domain of E.coli • Inhibited with red light• Requires phycocyanobilin (PCB)

• Covalently associated chromophore• Produced from heme by genes ho1 and

pcyA

PCB

resp. reg.OmpRphorphoryl

group

Sensor• Phytochrome– Membrane bound extracellular sensor/photoreceptor– Intracellular (response) regulator• Usu do not have DNA-binding domains• Do not directly regulate gene expression

• Fusion protein: phytochrome and EnvZ histidine kinase domain

Extracellular sensor

Intracellular regulator

Photoreceptor Expression

• PCB not produced in E.Coli• Introduction of PCB synthesis genes• Converts haem into PCB

Transduction Mechanism

• Red light represses EnvZ auto-phosphorylation

Output

• OmpR-P activates promoter linked to reporter gene

• lacZ reporter produce black compound

OmpR-P dependent ompC promoter

System Revisited

Light Canon

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