Difference between revisions of "Part:BBa K1583112"

Revision as of 15:00, 17 September 2015

pRha + CsgA & GFP in same operon

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. The aggregation to the nanowire ('curli') is induced by the membrane protein CsgB. CsgC, CsgE, CsgF and CsgG act as chaperones for CsgA during translation and export CsgA to the extracellular space.
This part was designed to measure intracellular expression rates of CsgA coupled to fluorescence (GFP) by cloning the biobrick BBa_I13504 into the same operon which is under control of the Rhamnose promoter.

Sequence and Features

A biofilm is created, when sufficient levels of mature CsgA are present in the extracellular space. CsgB can then act as a nucleator inducing the curli formation. On the way there, many questions need to be answered.
To do this in the most efficient way, we went back to good old modeling.

Transcriptional and translational rates can be approximated with reasonable precision. However, we wanted to measure this!</p>

To do so, we created this device. The beginning of the device consists of our standard parts: the rhamnose promoter and the CsgA gene. However, we skipped the terminator behind the gene.
In this manner we were able to clone the gene from the biobrick BBa_I13504 coding for GFP behind CsgA and into the same operon.
We assume that the difference between CsgA and CsgA+GFP in size does not influence transcription and translation of CsgA. Using a calibration curve connecting fluorescence [au] and mass [ng] we were able to calculate the expression rate of CsgA with units [proteins/(cell*second)].