IGEM:IMPERIAL/2007/Projects/In-Veso/Specifications

In-Veso Gene Expression: Specifications

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Characterization

Characterisation Chart

	Lifespan	Rate of Protein Synthesis	PoPS Effect	Membrane Traffic	Waste
Temperature
Media
Piping
Parts

Temperature

Range from 4-37^oC with priorities at:

1. 25^oC
2. 4^oC
3. 6-20^oC
4. 30-37^oC

Media

These are the different media the technique should be tested in, in order of priority:

1. Water
2. X-Gal
3. E. coli Biofilm
4. Agar Gel
5. Polyacrylamide
6. Paper

Tom 10:56, 26 July 2007 (EDT) Just a note to take into account Osmolarity of the media, which I'm sure you've thought of already! Also, B-Galactosidase is an enzyme not a type of media?

Piping

How narrow a pipe, and how fast, can the vesicles be passed through without bursting? Testing range..?

This is valuable for determining the spray system to be used in the Biofilm application. It also helps with laboratory manipulations - pipetting, etc.

Parts

List of parts from the registry we would like to test in-veso.

Protocol

Above are two choices for making cell extract from E.coli. One of them is S30, and the other is the S12 method, which is a more rapid and cost-effective preparation. The cell extracts can also be obtained from a manufacturer.

This is a protocol for obtaining the cell extract S30

The protocol is based on that of Karp and Pellinen.
1. E. coli A19 cells grown in SB medium (4× 500 ml) until on OD600 of 1.4
2. Cool cultures at 8 °C for 35 min
3. Harvest cells by centrifugation at 3000g for 15 min at 4 °C
4. Discard supernatant and wash pellets 3 times with 50 ml ice-cold S30 buffer
5. Store cells overnight at −72 °C

Overnight

6. Resuspend pellet in ice-cold S30 buffer (1.27 ml buffer/1 g cells)
7. Cells were lysed by one passage through a French press at 1300–1500 psi
8. Immediately add DDT to final concentration of 1 mM
9. Centrifuge lysate at 30,000g for 30 min
10. Transfer supernatant to a new tube to centrifuge again at 50,000g for 30 min
11. One milliliter of preincubation mix (see Table 1) was added to every 6.5 ml of S30 extract and then agitated for 60 min at RT. The solution was transferred to a dialysis tube (mol wt cutoff 6000–8000) and dialyzed three times against 50 vol of chilled S30 buffer at +8 °C. S30 buffer was replaced after every 1 h of incubation. Finally, the S30 extract was divided into aliquots to freeze in liquid nitrogen and to be stored at −72 °C.

Physical Conditions

temperature, pH, media, centrifugation