Difference between revisions of "Part:BBa K3765021"

Latest revision as of 20:16, 20 October 2021

OpdA+SpyTag002+sfGFP with OpdA signal peptide under T7 promoter

OpdA-SpyTag002-sfGFP fusion protein generator.

Usage and Biology

This protein generator can be used for expressing the OpdA-SpyTag002-sfGFP fusion protein under a strong bacteriophage T7 promoter by the T7 RNA Polymerase in suitable bacterial strains like E. coli BL21(DE3). Transcriptional regulation is by the lac operator sequence, and expression can be switched on via IPTG induction. Also contains a strong T7 RBS upstream and wild type T7 terminator downstream of the CDS for initiation and termination of translation.

Fig 1. Feature Map

To construct this protein generator, we cloned the OpdA-SpyTag002-sfGFP CDS into the high copy number pETMCN vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning instead of restriction digestion-ligation.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 2181
Illegal SpeI site found at 45
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2187
Illegal SpeI site found at 45
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2009
Illegal BamHI site found at 2169
Illegal XhoI site found at 2151
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 2181
Illegal SpeI site found at 45
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 2181
Illegal SpeI site found at 45
Illegal AgeI site found at 1308
Illegal AgeI site found at 1371
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 [[File:T--IISc-Bangalore--images--Map--C2--pETMCN-T7.png|thumb|center|800px|Fig 1. Feature Map]]
-To construct this protein generator, we cloned the [https://parts.igem.org/Part:BBa_K3765013 OpdA-SpyTag002-sfGFP CDS] into the high copy number [https://parts.igem.org/Part:BBa_K3765011 pETMCN] vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning insread of restriction digestion-ligation.
+To construct this protein generator, we cloned the [https://parts.igem.org/Part:BBa_K3765013 OpdA-SpyTag002-sfGFP CDS] into the high copy number [https://parts.igem.org/Part:BBa_K3765011 pETMCN] vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning instead of restriction digestion-ligation.
 ----