Difference between revisions of "Part:BBa K1418002"

Revision as of 21:19, 1 November 2014

Chlorophyllase (TaCHL) generator with C-terminal 10x His tag

TaCHL is a chlorophyllase enzyme from Triticum aestivum, a common species of wheat. Primers were designed to use polymerase chain reaction (PCR) to amplify the TaCHL open reading frame from the pET28a vector that the gene was provided in (Dr. Joseph Jez, Washington University, St. Louis, MO). Since we were using the chlorophyllase gene in gene fusion applications, RFC 23 sequences were incorporated into the primers used for amplification. The amplified product was cloned into pSB1C3 and designated as BBa_K1418000. Primers were then designed and PCR was performed to remove the stop codon from TaCHL. This amplified product was cloned into pSB1C3 and is designated as BBa_K1418000.Primers were then designed and PCR was performed to remove the stop codon from TaCHL. This amplified product was cloned into pSB1C3 and is designated as BBa_K1418001. By removing the stop codon, in frame fusions on the 3' end of the gene can be performed. Part BBa_K1418001 was then cloned behind a lac inducible promoter and a ribosome binding site (K208010 contains both R0010 and B0034). To aid in protein purification, a 10x histidine tag with two transcriptional terminators was cloned in frame on the 3' end to generate the final composite construct, BBa_K1418002.

To test for protein production from the new BBa_K1418002 construct, protein purification using a nickel column was performed. Using methods provided in our "protocols" section, cells containing BBa_K1418002 were grown overnight, pelleted and lysed. After centrifugation, the supernatant was applied to the nickel column. Various samples throughout the purification process were analyzed using SDS-PAGE. The SDS-PAGE below shows results from analysis of cells containing BBa_K1418002.

It can clearly be seen from the SDS-PAGE that a protein product between 25kDa and 37kDa has been purified using the nickel column. Since the expected size of our chlorophyllase construct is 34 kDa, we are confident that we have purified the chlorophyllase protein.

To demonstrate that our chlorophyllase enzyme that we produced had function, we ran an assay that would visually show the change from a hydrophobic chlorophyll molecule (in the organic phase) to a hydrophilic head (in the aqueous phase) that was a result of the chlorophyllase cleaving the phytol tail from the chlorophyll molecule.

To further characterize our part and show that this BioBrick-produced enzyme will help remove chlorophyll from clothing, we first cut cotton strips from a lab coat. We then "stained" the cloth with commercial chlorophyllase, allowed it to dry, and treated it with our purified chlorophyllase for 5 minutes. Both the control (buffer alone) and the treated (buffer and chlorophyllase) were then rinsed briefly with water to allow for removal of any water soluble molecules. Seen in the image below, the treated cotton strip showed a greatly reduced stain.

Again, to further characterize our part and show that this BioBrick-produced enzyme will help remove actual stains from clothing, we took more cotton strips and agitated them grass to create a grass stain. After approximately 30 minutes of treatement, both the control (buffer alone) and the treated (buffer and chlorophyllase) were then rinsed briefly with water to allow for removal of any water soluble molecules. In the image below, the treated cotton strip showed a greatly reduced stain and removal of a majority of the green pigment (chlorophyll).

Sequence and Features