Difference between revisions of "Part:BBa K1418000"

Revision as of 03:44, 18 October 2014

Chlorophyllase (TaCHL from Triticum aestivum)

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. 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!

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 885
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 43
Illegal NgoMIV site found at 424
Illegal NgoMIV site found at 523
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 905

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1418000 short</partinfo>
-TaCHL is a chlorophyllase enzyme from <i>Triticum aestivum</i>, 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.  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.
+TaCHL is a chlorophyllase enzyme from <i>Triticum aestivum</i>, 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.  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.
+https://static.igem.org/mediawiki/2014/e/e1/2014USU_K1418002PlasmidPic.png
+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.
+https://static.igem.org/mediawiki/2014/8/8f/2014USU_AnnotatedTaCHLProteinGel.png
+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!
 <!-- Add more about the biology of this part here