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

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In-Veso Gene Expression: Notes



Shopping List

  1. L1020 E.coli S30 extract system for circular DNA; Promega; Quantity: 1
  2. L1130 E.coli T7 S30 extract system for circular DNA; Promega; Quantity: 1

# R9379 Rhodamine B isothicynate-Dextran; Sigma; Quantity: 1 # Fluorescein-12-dUTP; Serial no. 11373242910; Roche; Quantity: 1

  1. P9511 3-sn-Phosphatidic acid sodium salt from egg yolk lecithin; Sigma; Quantity: 1
  2. M5904 Mineral oil, Sigma, Quantity: 500ml
  3. 42492 1,2-Dioleoyl-sn-glycero-3-phosphocholine. Quantity: 5ml
  4. LAA21 L-amino acids. Sigma. Quantity: 1
  5. R0884 T7 RNA Polymerase. Sigma. Quantity: 1 100mg/ml
  6. 43817 DL Dithiothreitol. Fluka. Quantity: 5g
  7. FLAAS Adenosine 5 triphosphate (ATP) disodium salt hydrate. Sigma. Quantity: 5vl
  8. 860077 Phopho(enol)pyruvic acid monopotassium salt. Aldirch. Quantity: 1g
  9. P1903 Pyruvate Kinase from Bacillus stearothermophilus. Sigma. Quantity: 1
  10. I6758 Isopropyl b D 1 thiogalactopyranoside. Sigma. Quantity: 5g
  11. 63052 Magnesium acetate solution. Fluka. Quantity: 500ml
  12. G1501 L-Glutamic acid potassium salt monohydrate. Sigma. Quantity: 500g
  13. M6250 2-Mercaptoethanol. Sigma. Quantity: 100ml
  14. Y2377 2X YT Microbial Medium. Sigma. Quantity:250g
  15. B2685 BL21 Competent Cells, Uni-pack. Quantity: 1

Extras

  1. 362794 Cholesterol. Aldrich. Quantity: 5g

Revised Shopping List

  1. L1020 E.coli S30 extract system for circular DNA; Promega; Quantity: 1
  2. L1130 E.coli T7 S30 extract system for circular DNA; Promega; Quantity: 1
  3. P9511 3-sn-Phosphatidic acid sodium salt from egg yolk lecithin; Sigma; Quantity: 1
  4. M5904 Mineral oil, Sigma, Quantity: 500ml
  5. 42492 1,2-Dioleoyl-sn-glycero-3-phosphocholine. Quantity: 5ml

Timeline

Week 4


Mon: Order reagents
Tue: -
Wed: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract
Thur: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract
Fri: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract


Week 5


Mon: Test if S30 or S12 is better for the respective strains of E.coli
Tue: Carry out temperature and pH tests on in vitro and in veso extracts
Wed: Carry out temperature and pH tests on in vitro and in veso extracts
Thur: Carry out temperature and pH tests on in vitro and in veso extracts
Fri: Carry out temperature and pH tests on in vitro and in veso extracts

Proposal for Lab

We will first work on making gene constructs and cloning them in E.coli. Restriction digestion, ligation and transformation will be used. Agarose gel electrophoresis will then be used to test for products formed. Cell extracts will be bought from Molecular biology suppliers. Miniprep will be performed to extract plasmids from the cells. The plasmids will then be used to express proteins in vitro and synthethic vesicles, and the proteins tested for by using a fluorometer. Vesicles will be made with phospholipids bought from a supplier. We also need to test these systems for their temperature and pH ranges, as well as their lifespans. All these will use standard equipment in the lab.

On α-Hemolysin

There are different kinds of nanopores used for different applications - both organic, such as HlyA (α-Hemolysin), and synthetic, solid-state pores produced primarily by elctron-beam-assisted techniques.[1] READ MORE

The composition of the lipid bi-layer membrane affects its stability when HlyA pores are inserted. Namely, adding the positive curvature agent lysophosphatidylcholine significantly increases the stability of the membrane. Untreated membranes had their lifetime decreased by up to three orders of magnitude[2] READ MORE

However, the insertion of HlyA does not necessarily cause lysis - different variants of the protein have different effects, and varying insertion rates. This has implications to membrane stability - a non-lysing variant may be preferred. Further, HlyA requires the presence of Calcium for insertion.[3]

An important group of OM (outer membrane) channels are the porins. Of these, OmpG is unique in that it has a large (approximately 13 A) central pore that is very similar in size to that of the toxin α-Hemolysin. OmpG may form a non-specific channel for the transport of larger oligosaccharides. The structure of OmpG provides the starting point for engineering studies aiming to generate selective channels and for the development of biosensors.[4] READ MORE

No E.coli K-12 strain tested carries the HlyA gene. However, the silent α-Hemolysin gene, sheA, is present in all K-12 strains tested.[5]


  1. Rhee M and Burns MA. Nanopore sequencing technology: nanopore preparations. Trends Biotechnol. 2007 Apr;25(4):174-81. DOI:10.1016/j.tibtech.2007.02.008 | PubMed ID:17320228 | HubMed [1]
  2. Bakás L, Chanturiya A, Herlax V, and Zimmerberg J. Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes. Biophys J. 2006 Nov 15;91(10):3748-55. DOI:10.1529/biophysj.106.090019 | PubMed ID:16935953 | HubMed [2]
  3. Sánchez-Magraner L, Cortajarena AL, Goñi FM, and Ostolaza H. Membrane insertion of Escherichia coli alpha-hemolysin is independent from membrane lysis. J Biol Chem. 2006 Mar 3;281(9):5461-7. DOI:10.1074/jbc.M512897200 | PubMed ID:16377616 | HubMed [3]
  4. Subbarao GV and van den Berg B. Crystal structure of the monomeric porin OmpG. J Mol Biol. 2006 Jul 21;360(4):750-9. DOI:10.1016/j.jmb.2006.05.045 | PubMed ID:16797588 | HubMed [4]
  5. Kerényi M, Allison HE, Bátai I, Sonnevend A, Emödy L, Plaveczky N, and Pál T. Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains. J Clin Microbiol. 2005 Jun;43(6):2965-8. DOI:10.1128/JCM.43.6.2965-2968.2005 | PubMed ID:15956433 | HubMed [5]

All Medline abstracts: PubMed | HubMed

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