Difference between revisions of "Part:BBa J100091"
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'''J100091''' was built by Todd Eckdahl and allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for [http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate Golden Gate Assembly] using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see [[Part:BBa_J119022]] as an example or the picture below). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. [[Part:BBa_J100091]] and [[Part:BBa_J119044]] incorporate the BD18 bicistronic translational junction (see [[Part:BBa_J119024]]) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.  
 
'''J100091''' was built by Todd Eckdahl and allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for [http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate Golden Gate Assembly] using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see [[Part:BBa_J119022]] as an example or the picture below). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. [[Part:BBa_J100091]] and [[Part:BBa_J119044]] incorporate the BD18 bicistronic translational junction (see [[Part:BBa_J119024]]) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.  
  
−
Below is a picture of what this part will look like when digested with Bsa I. TT represents the transcriptional terminator [[Part:BBa_B0014]]. This part is designed to be used for Golden Gate Assembly to swap out the TT for a promoter of your choosing. This can be done by simply mixing [[Part:BBa_J100091]]  with oligos that [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html self-assemble into dsDNA with compatible sticky ends.] [[Part:BBa_J100091]] and its sister part [[Part:BBa_J119044]] are ideal for undergraduates in a teaching lab to [http://gcat.davidson.edu/RFP/ discover and characterize new promoters for use in synthetic biology].  
+
Below is a picture of the portion that pops out when digested with Bsa I. TT represents the transcriptional terminator [[Part:BBa_B0014]]. This part is designed to be used for Golden Gate Assembly to swap out the TT for a promoter of your choosing. This can be done by simply mixing [[Part:BBa_J100091]]  with oligos that [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html self-assemble into dsDNA with compatible sticky ends.] [[Part:BBa_J100091]] and its sister part [[Part:BBa_J119044]] are ideal for undergraduates in a teaching lab to [http://gcat.davidson.edu/RFP/ discover and characterize new promoters for use in synthetic biology].  
  
 
[http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate_Assembly_protocol See the full protocol.]<br><center>
 
[http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate_Assembly_protocol See the full protocol.]<br><center>

Revision as of 13:53, 20 August 2012

For Testing New Promoters via Golden Gate Assembly

J100091 was built by Todd Eckdahl and allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for [http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate Golden Gate Assembly] using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see Part:BBa_J119022 as an example or the picture below). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. Part:BBa_J100091 and Part:BBa_J119044 incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.

Below is a picture of the portion that pops out when digested with Bsa I. TT represents the transcriptional terminator Part:BBa_B0014. This part is designed to be used for Golden Gate Assembly to swap out the TT for a promoter of your choosing. This can be done by simply mixing Part:BBa_J100091 with oligos that [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html self-assemble into dsDNA with compatible sticky ends.] Part:BBa_J100091 and its sister part Part:BBa_J119044 are ideal for undergraduates in a teaching lab to [http://gcat.davidson.edu/RFP/ discover and characterize new promoters for use in synthetic biology].

[http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate_Assembly_protocol See the full protocol.]

J100028.png

J100091 contains BBa biobrick prefix + BbsI site + 4 base overhang + BsaI site cutting to the left + transcriptional terminator Part:BBa_B0014 + BsaI site that cuts to the right + 4 base overhang + BbsI site + BD18 bicistronic RBS for more reliable translation (Part:BBa_J119024) + mRFP Part:BBa_E1010 + BBa biobrick suffix.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 857
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 858
    Illegal PstI site found at 872
    Illegal NotI site found at 7
    Illegal NotI site found at 865
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 858
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 858
    Illegal PstI site found at 872
    Illegal AgeI site found at 730
    Illegal AgeI site found at 842
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 135
    Illegal BsaI.rc site found at 34


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 906
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal NotI site found at 7
    Illegal NotI site found at 914
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal AgeI site found at 779
    Illegal AgeI site found at 891
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 129
    Illegal BsaI.rc site found at 28