BioSketch BioBricks Reporters

From FreeBio

GFP

We had originally planned to use GFP as a test reporter. However, the part that we ordered from the Registry never arrived, so this was abandoned.

EYFP

EYFP (BBa_E0430) was obtained for BioWire. This replaced GFP as a test reporter for the BioSketch.

So far, we have a good success with this reporter. It has been visualized with the LacI, Lambda cI, and 434 cI regulated promoters infront of it.

The orignal intent was to use this reporter to confirm that the promoters were working and then replace it with mCherry. However, the yeast mCherry from the Registry has encountered some problems (see below). So, we are proceeding with EYFP as our final reporter, but are also hoping that the bacterial mCherry will be ready in time.

Yeast mCherry

This was orignally supposed to be our final reporter. The advantage of using mCherry as a reporter is that it is visible under regular light, thus removing the need for special equipment for our final product. The yeast mCherry (BBa_E2060) came in the KAN plasmid and due to read-through, the cells were red/pink, especially when pelleted.

However, when the terminator was added, the cells no longer exhibited the red color. This was not completely unexpected, since the construct was in the AMP plasmid at this point. So, construction continued with the addition of the RBS.

Adding promoters in front of the RBS-mCherry-T construct did not cause the cells to have color. The construct was then sent out for sequencing twice and both returned a DNA sequence that had little resemblance to the desired construct.

Given the late date and the fact that the bacterial mCherry was progressing well, the yeast mCherry has been abandoned.

Yeast Venus YFP

This was orignally a secondary reporter to test against the yeast mCherry. However, the yeast Venus YFP (BBa_E2030) encountered the same problems as the yeast mCherry and has likewise been abandoned.

Bacterial mCherry

Bacterial mCherry was obtained from Roger Y. Tsien's lab. Using PCR, a PstI site was removed and BioBrick ends were added. The Bacterial mCherry was created in a plain (BBa_J06504) and with LVA added (BBa_J06505). Both were successfully created and had their sequences confirmed.

We are currently adding on the RBS and terminator and are hoping to have it ready in time to add it to our final constructs. Nevertheless, we are proceeding with EYFP as our reporter.