Difference between revisions of "Part:BBa K782007"
UrosZupancic (Talk | contribs) |
(→Characterization) |
||
| (9 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K782007 short</partinfo> | <partinfo>BBa_K782007 short</partinfo> | ||
| + | |||
==Introduction== | ==Introduction== | ||
| Line 11: | Line 12: | ||
Single DNA binding sequence for NicTAL:TCTATCAATGATAGA | Single DNA binding sequence for NicTAL:TCTATCAATGATAGA | ||
| + | |||
==Characterization== | ==Characterization== | ||
| − | We found out [https://parts.igem.org/Part:BBa_K323214 | + | We found out that [https://parts.igem.org/Part:BBa_K323214 NicTAL10 ] part deposited in the Registry by the Slovenian iGEM2010 team is not functional. Part was missing subdomain in DNA-binding domain, so [http://2012.igem.org/Team:Slovenia our team] added the missing subdomain on the N terminus, since at that time the requirement for the minimal DNA binding domain has not been known. Apart from adding the missing N terminal aminoacids, we added a HIS tag also on the N terminus and NLS on the C terminus of NicTAL12 (Figure 2). This construct was later for designing TAL-based activator and repressor that were experimentaly verified by adding [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782066 VP16] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 KRAB] domain. |
[[Image:Svn_12_NicTAL10vs12.PNG | 600 px]] | [[Image:Svn_12_NicTAL10vs12.PNG | 600 px]] | ||
| Line 21: | Line 23: | ||
| − | |||
| − | |||
[[Image:Svn_12_NicTAL_graf.png]] | [[Image:Svn_12_NicTAL_graf.png]] | ||
| − | '''Figure 3:''' Results | + | '''Figure 3:''' Results obtained by testing [https://parts.igem.org/Part:BBa_K782011 NicTAL12:KRAB] repressor proved better binding ability since NicTAL12 gave much better results than NicTAL10:KRAB. HEK293T cells were cotransfected with [https://parts.igem.org/Part:BBa_K782011 NicTAL12:KRAB] repressor under the control of a CMV promoter, and with a firefly luciferase reporter plasmid [https://parts.igem.org/Part:BBa_K782023 (BBa_K782023)] containing 12 DNA-binding sites for NicTAL repressor upstream the CMV promoter. Check detailed results on [http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators iGEM 2012 team Slovenia wiki]. |
==References== | ==References== | ||
| Line 34: | Line 34: | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
| + | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 00:04, 27 September 2012
NicTAL12:NLS DNA binding domain
Introduction
TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).
Figure 1: Schematic representation of the construct.
Single DNA binding sequence for NicTAL:TCTATCAATGATAGA
Characterization
We found out that NicTAL10 part deposited in the Registry by the Slovenian iGEM2010 team is not functional. Part was missing subdomain in DNA-binding domain, so [http://2012.igem.org/Team:Slovenia our team] added the missing subdomain on the N terminus, since at that time the requirement for the minimal DNA binding domain has not been known. Apart from adding the missing N terminal aminoacids, we added a HIS tag also on the N terminus and NLS on the C terminus of NicTAL12 (Figure 2). This construct was later for designing TAL-based activator and repressor that were experimentaly verified by adding VP16 and KRAB domain.
Figure 2: Sequence alignment of NicTAL10 and NicTAL12 showing differences between constructs.
Figure 3: Results obtained by testing NicTAL12:KRAB repressor proved better binding ability since NicTAL12 gave much better results than NicTAL10:KRAB. HEK293T cells were cotransfected with NicTAL12:KRAB repressor under the control of a CMV promoter, and with a firefly luciferase reporter plasmid (BBa_K782023) containing 12 DNA-binding sites for NicTAL repressor upstream the CMV promoter. Check detailed results on [http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators iGEM 2012 team Slovenia wiki].
References
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2133
Illegal XhoI site found at 1224 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]