Monday, August 13

Crocin Extraction

To accurately verify that our construct is indeed producing zeaxanthin, spectrophotometry must be used to characterize our biobrick part. To this end, we tested crocin in two different solvents, along with our Z^2 construct, compared to the absorption spectrum of zeaxanthin in hexane.

http://dl.dropbox.com/u/88390549/CrocinGraphs.png

This indicates that our Z^2 construct isn't actually producing crocin, or if it is, the crocin isn't expressed highly enough to absorb significantly.

Saffron in a Kan

We also PCR'd the Z construct in the submission plasmid, Psb1c3. We then digested the results with both EcoRI and PstI, as shown below, and saw that the PCR had indeed succeded.

Finally, we ran an SDS-PAGE gel of our Z construct with a histidine tag in order to see the proteins produced using an extraction protocol from Sigma found here [INSERT PROTOCOL].
Tuesday, August 14

Performed another extraction using His-columns to purify ZCD with His tag and UGTCS2 with His tag and ran them on a SDS-Page gel.

PCR of TOPO with Z and U again using colonies as template.

In vitro assay for crocin production repeated with purified protein from extraction mentioned above.

Digest of PsbA2 PCR'd with ZCD and control TOPO vector - cut with E and P Ligation of the above products with the chloramphenicol plasmid.

Wednesday, August 15

Transformation of the ligation yesterday using GC10s and 5 microliters of ligation mixture.

PCR of Z + Thiofusion at three different temperatures (45, 50, and 55 degrees) in order to figure out why the PCR isn't working.

We also attempted to perform a zeaxanthin extraction using our culture of the Synechocystis PAL mutant. [EXTRACTION PICTURE]

Thursday, August 16

Friday, August 17