Difference between revisions of "Part:BBa K2918016"

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===Usage and Biology===
 
===Usage and Biology===
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A series of T7 terminator variants were generated by randomized mutagenesis (Temme et al, 2012). These variants can be used when assembling multiple transcriptional units to reduce chances of homologous recombination between similar sequences.  
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A series of T7 terminator variants were generated by randomized mutagenesis <html><a href="#Temme2012">(Temme et al., 2012)</a></html>. These variants can be used when assembling multiple transcriptional units to reduce chances of homologous recombination between similar sequences.
  
 
===Strain Construction===
 
===Strain Construction===
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The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone [http://www.addgene.org/47994 pICH41276] and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
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The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone <html><a href="http://www.addgene.org/47994"> pICH41276 </a></html> and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
  
 
===Modular Cloning===
 
===Modular Cloning===
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<b>Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html></b>
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<b>Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. </b>
  
  
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===References===
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<a id="Temme2012" href="https://pubs.acs.org/doi/10.1021/acssynbio.7b00258" target="_blank">
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Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F., & Voigt, C. A. (2012). Modular control of multiple pathways using engineered orthogonal T7 polymerases. <i>Nucleic acids research</i>, 40(17), 8773–8781.</a>
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Latest revision as of 18:12, 6 December 2019

T7 terminator variant

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]

Usage and Biology

A series of T7 terminator variants were generated by randomized mutagenesis (Temme et al., 2012). These variants can be used when assembling multiple transcriptional units to reduce chances of homologous recombination between similar sequences.

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone pICH41276 and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.


Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2.


Table 1: Overview of different level in MoClo

Level Basic/Composite Type Enzyme
Level 0 Basic Promoters, 5’ UTR, CDS and terminators BpiI
Level 1 Composite Transcriptional units BsaI
Level 2/M/P Composite Multiple transcriptional units BpiI

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.


Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Basic Part Sequence 5' End Sequence 3' End Level 0 backbone
Promoter NNNN GAAGAC NN GGAG TACT NN GTCTTC NNNN pICH41233
5’ UTR NNNN GAAGAC NN TACT AATG NN GTCTTC NNNN pICH41246
CDS NNNN GAAGAC NN AATG GCTT NN GTCTTC NNNN pICH41308
Terminator NNNN GAAGAC NN GCTT CGCT NN GTCTTC NNNN pICH41276

References