Difference between revisions of "Part:BBa K1639015"
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===Usage and Biology=== | ===Usage and Biology=== | ||
'''Cloning into pTet-off''' | '''Cloning into pTet-off''' | ||
− | This part contains VP16 activator complex with appropriate[[File: | + | This part contains VP16 activator complex with appropriate[[File:pTet off-373.png|right|360px|thumb|'''Figure 1:'''Schematic diagram of cloning into pTet-off]] enzyme cut sites and miRNA binding sites for high-miRNas. Originally it's same with VP16 part of tetracycline-controlled transactivator (tTA), comprising a fusion of the '''tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16''' By including Sal1 cut site at the beginning of this part we can change it with original tTA part found in pTet-off plasmid. ''(Figure 1)'' We performed ligation and then transformed the plasmids into BL21 bacteria. After 16 hours incubation at 37 C, we performed colony PCR and cut-check to check for positive clones. Despite trying this strategy 7 or 8 times, we were unable to successfully clone this g-block into pTET off. <br clea=all> |
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+ | Below there is a whole diagram of Cancer module. This part is first step in this machinery. ''(Figure 2)'' | ||
+ | [[File:ATOMS-Turkiye_cancer_switch_1.1.png|center|450px|thumb|'''Figure 2''']] | ||
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Latest revision as of 23:15, 21 September 2015
VP16 Transcriptional Activation Domain with miR373 Binding Site
Usage and Biology
Cloning into pTet-off
This part contains VP16 activator complex with appropriate enzyme cut sites and miRNA binding sites for high-miRNas. Originally it's same with VP16 part of tetracycline-controlled transactivator (tTA), comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16 By including Sal1 cut site at the beginning of this part we can change it with original tTA part found in pTet-off plasmid. (Figure 1) We performed ligation and then transformed the plasmids into BL21 bacteria. After 16 hours incubation at 37 C, we performed colony PCR and cut-check to check for positive clones. Despite trying this strategy 7 or 8 times, we were unable to successfully clone this g-block into pTET off.Below there is a whole diagram of Cancer module. This part is first step in this machinery. (Figure 2)
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 335
Illegal PstI site found at 341 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 335
Illegal PstI site found at 341
Illegal NotI site found at 327 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 335
Illegal PstI site found at 341 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 335
Illegal PstI site found at 341 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 320