Difference between revisions of "Part:BBa K2589001"

Latest revision as of 01:14, 18 October 2018

Short His J-Protein (trc-promoter)

This part is a composite of the 2017 WLC-iGEM lambda phage Short J-Protein with N-Terminal His tag (BBa_K2452002) downstream of the trc-promoter (BBa_K2042004) allowing for Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction of the coding region.

Characterization of Part Improvement

The 2017 iGEM Team had performed some characterization experiments of the lambda phage short J-protein with N-Terminal His tag under the control of the trc-promoter without submitting a part containing either the trc-promoter BBa_K2042004 or a composite part with pTrc99A regulatory elements. In addition, this data was never entered on the registry to characterize any parts when it should have been submitted as characterization for previous parts. As a result, we are submitting this data used with permission from the 2017 WLC-Milwaukee Team as characterization of BBa_K2589001 protein expression and purification when induced or uninduced. This data is the result of a native SDS PAGE Gel electrophoresis stained using standard Coomassie staining methods. Protein expression is seen to be significantly improved under IPTG induction conditions, although purification by Nickel Column His tag affinity seems to be unsuccessful as there is protein in the flow through samples.

To ensure the presence of the His tag on BBa_K2452002 (as the affinity purification seemed unsuccessful) Western Blot analysis was performed seen below confirming that the protein is expressed with the His tag.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2589001 short</partinfo>
-This part is a composite of the 2017 WLC-iGEM lambda phage Short J-Protein with N-Terminal His tag (BBa_K2452002) downstream of the trc-promoter (derived from pTrc99a) allowing for Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) induction of the coding region.
+This part is a composite of the 2017 WLC-iGEM lambda phage Short J-Protein with N-Terminal His tag (BBa_K2452002) downstream of the trc-promoter (BBa_K2042004) allowing for Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) induction of the coding region.
-===Characterization===
+===Characterization of Part Improvement===
+The 2017 iGEM Team had performed some characterization experiments of the lambda phage short J-protein with N-Terminal His tag under the control of the trc-promoter without submitting a part containing either the trc-promoter BBa_K2042004 or a composite part with pTrc99A regulatory elements. In addition, this data was never entered on the registry to characterize any parts when it should have been submitted as characterization for previous parts. As a result, we are submitting this data used with permission from the 2017 WLC-Milwaukee Team as characterization of BBa_K2589001 protein expression and purification when induced or uninduced. This data is the result of a native SDS PAGE Gel electrophoresis stained using standard Coomassie staining methods. Protein expression is seen to be significantly improved under IPTG induction conditions, although purification by Nickel Column His tag affinity seems to be unsuccessful as there is protein in the flow through samples.
 https://static.igem.org/mediawiki/2017/6/67/T--WLC-Milwaukee--Results_SDS_page_2_Med.jpg
+To ensure the presence of the His tag on BBa_K2452002 (as the affinity purification seemed unsuccessful) Western Blot analysis was performed seen below confirming that the protein is expressed with the His tag.
+https://static.igem.org/mediawiki/2017/7/72/T--WLC-Milwaukee--Results_Western_1_Med.jpg
 <!-- Add more about the biology of this part here