IGEM:IMPERIAL/2007/Projects/In-Veso/Specifications
In-Veso Gene Expression: Specifications
Characterization
Resources
IC Specialists
Literature
Wetlab
Characterisation Chart
Lifespan | Rate of Protein Synthesis |
PoPS Effect | Protocol | Membrane Traffic | Waste | |
Temperature | ||||||
Media | ||||||
Piping | ||||||
Parts |
Temperature
Range from 4-37oC with priorities at:
- 25oC
- 4oC
- 6-20oC
- 30-37oC
Media
These are the different media the technique should be tested in, in order of priority:
- Water
- Agar Gel
- β-Galactosidase
- Polyacrylamide
- Paper
- E. coli Biofilm
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. Then the pellet was resuspended in ice-cold S30 buffer (1.27 ml buffer/1 g cells) (see Table 1). Cells were lysed by one passage through a French press (SLM Aminco, SLM Industries) at 1300–1500 psi. DTT was added immediately to final concentration of 1 mM. Then the lysate was centrifuged at 30,000g for 30 min and supernatant transferred to a new tube to centrifuge again at 50,000g for 30 min. 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.
Rate of PoPs
Comparison with the efficiency of other chassis
Limitations of type of proteins made in the vesicles
Life span
Membrane Permeability
Physical Conditions
temperature, pH, media, centrifugation