Composite

Part:BBa_K1583203

Designed by: Stefan Robert Marsden   Group: iGEM15_TU_Delft   (2015-09-04)
Revision as of 14:34, 17 September 2015 by SMarsden (Talk | contribs)

Low constitutive promoter + CsgB & CsgC

CsgB functions as the anchor on the cell wall for curli nanowires. It triggers the aggregation of CsgA and leads to the formation of amyloid nanowires. CsgC is positively involved in the extracellular aggregation of CsgA into amyloid nanowires (curli assembly) in E.coli functioning as a chaperone for CsgA. Constructs with different promoter strenghts were designed to investigate the impact on nanowire self-assembly.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

A bacterial biofilm consists up to 80% of amyloid nanowires. These nanowires are often called curlis and they are made from a protein monomer called CsgA. Hundreds and thousands of these monomers aggregate to a form a nanowire of up to several micrometers in length. The curli nucleation process to is induced by the membrane protein CsgB, which functions as a cellular anchor via which the nanowire attaches to the cell.

Our modeling results showed, that the amount of CsgB distributed over the bacterial cell wall has a great impact on the properties of the biofilm: whereas a high amount of CsgB leads to many, but short curlis, only few CsgB proteins lead to long nanowires connecting the cells. In order to investigate this more thoroughly, this device was designed.

CsgC has the function to prevent intracellular aggregation of CsgA and thus the formation of nanowires within the cell. Along with other proteins
(CsgE, CsgF, CsgG) CsgC is an important parameter in providing mature CsgA in the extracellular space in the most efficient way.

But which is the most efficient way?

By creating devices in which the protein CsgC is constitutively expressed at different levels using three different promoter strengths, we were hoping to identify the expression level corresponding to the strongest biofilm.

Relative promoter strengths [au]:

J23110: 844

J23118: 1429

J23100: 2547

This device was intended as a platform for the further addition of CsgE, CsgF and CsgG genes to investigate their impact on the amount of available, mature, extracellular CsgA.
Since this device contains CsgB, exported CsgA will ultimately nucleate and start creating curlis from CsgB embedded in the cell wall.


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