Part:BBa_K426020

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 a variety of medias, including LB, TB, artificial sea water (ASW), and Cereal Grass Media(CGM) at different ratios. Here is a graph of our TECAN data from some of those Self-Lysis Tests:

This data shows that the self lysis device works best in TB, fairly well in CGM and not at all in ASW. For our payload delivery scheme, we decided to use CGM because in addition to allowing lysis, it's also a healthy media for Choanoflagellates.

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