Composite

Part:BBa_K426020

Designed by: Daniela Mehech   Group: iGEM10_Berkeley   (2010-10-27)
Revision as of 17:57, 27 October 2010 by Registry (Talk | contribs)

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Self-Lysis Device under Pbad and with BRP

The Self Lysis Device acts to degrade the bacterial membrane (inner membrane, peptidoglycan layer and outer membrane).

The Construct

Pbad promoter: allows for fast, controlled induction, allows better control of the timing of lysis, addresses biosafety concerns since this promoter is only induced by an exogenous molecule.

Bacteriocin Releasing Protein (BRP): degrades the outer membrane

Lysozyme: degrades the peptidoglycan layer

Holin: creates pores in the inner membrane

Anti-holin: threshold-gating system

(for more complete descriptions, see 2008 UC Berkeley iGEM team's wiki)

A variation of this part (that does not include BRP) was characterized in standard E. coli media, Luria Broth (LB), by the 2008 UC Berkeley iGEM team. In the Anderson lab, the construct was modified to include the BRP protein and has been extensively tested as a means of delivery in Mammalian cells. The 2010 Berkeley iGEM team, characterized the device in an entirely new setting: for use inside of Choanoflagellates.

Self-lysis can be assayed by measuring changes in optical density, or changes in the opaqueness of a solution. As lysis occurs, optical density decreases, meaning the solution of bacteria becomes more translucent. A TECAN machine was used to measure the change in optical density over time. The self-lysis device was tested in LB, TB, artificial sea water (ASW), and Cereal Grass Media(CGM). Tests showed that TB is the best media for lysis, followed by LB, CGM, high ratios of LB:ASW, low ratios of LB: ASW, and ASW. Also, macroscopic tests indicate that lysis occurs in spring water:LB ratios as high as 80:20.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2410
    Illegal NheI site found at 2433
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2795
    Illegal AgeI site found at 1028
    Illegal AgeI site found at 1396
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1010


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