Difference between revisions of "Part:BBa K2918011"
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===Usage and Biology===
 
===Usage and Biology===
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The (<a target=”_blank” href=”https://parts.igem.org/Part:BBa_K2918034”>broad host range promoter </a>) has been designed by combining promoter regions of <i>E.coli</i> , <i> B.subtilis </i> and <i> S.cerevisiae </i>. The promoter is based on the Pmin minimal promoter of S.cerevisiae.  It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in <i> E.coli</i> and <i>B.subtilis </i>. Therefore, to make the Pmin promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the Pmin promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′. <br>
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The <html><a href="https://parts.igem.org/Part:BBa_K2918000">broad host range promoter (P<sub>BHR</sub>)</a></html> has been designed by combining promoter regions of <i>E. coli</i>, <i> B. subtilis</i> and <i>S. cerevisiae</i>. The promoter is based on the P<sub>min</sub> minimal promoter of <i>S. cerevisiae</i>.  It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in <i> E. coli</i> and <i>B. subtilis </i>. Therefore, to make the P<sub>min</sub> promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the P<sub>min</sub> promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′. <br>
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The (<a target=”_blank” href=”https://parts.igem.org/Part:BBa_K2918034”>broad host range promoter </a>) was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). The 17bp spacer between the -35 and -10 conserved sequences (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) was replaced with a binding site for TALE protein. TALE proteins consist of repeats where 12th and 13th amino acids can vary, the repeats are called the repeat variable diresidue (RVD) (Segall-Shapiro, Sontag et al. 2018). These RVDs have been shown to bind to DNA in a simple one-to-one binding code. A unique 18bp binding site was incorporated into the promoter, this binding site recruits a specific TALE protein called TALEsp1 that acts as a repressor (Segall-Shapiro, Sontag et al. 2018). The TALEsp1 protein was designed to bind protein specifically at the binding site and are predicted not be bind anywhere in the genome (Segall-Shapiro, Sontag et al. 2018).
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The <html><a href="https://parts.igem.org/Part:BBa_K2918000">broad host range promoter (P<sub>BHR</sub>)</a></html> was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). The 17bp spacer between the -35 and -10 conserved sequences (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) was replaced with a binding site (16bp) for TALE protein. TALE proteins consist of repeats where 12th and 13th amino acids can vary, the repeats are called the repeat variable diresidue (RVD) <html><a href="#Segall2018">(Segall-Shapiro et al., 2018)</a></html>. These RVDs have been shown to bind to DNA in a simple one-to-one binding code. A unique 16bp binding site was incorporated into the promoter, this binding site recruits a specific TALE protein called TALEsp1 that acts as a repressor <html><a href="#Segall2018">(Segall-Shapiro et al., 2018)</a></html>. The TALEsp1 protein was designed to bind protein specifically at the binding site <html><a href="#Segall2018">(Segall-Shapiro et al., 2018)</a></html>.
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===Strain Construction===
 
===Strain Construction===
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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 <html><a href="http://www.addgene.org/47984">pICH41233</a></html> and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
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Aim: To clone the promoter in a level 0 MoClo backbone <html> (<a target=”_blank” href=”https://www.addgene.org/47984/”>pICH41233</a>) </html>.
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Procedure: The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible promoter overhangs. The cloning protocol can be found in the MoClo section below.
+
  
 
===Modular Cloning===
 
===Modular Cloning===
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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).
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Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments <html><a href="#Weber2011">(Weber et al., 2011)</a></html>. 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).
 
Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a> </html> for the protocol.  
 
Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a> </html> for the protocol.  
  
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     </body>
 
     </body>
 
</html>
 
</html>
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===Characterization===
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<div><ul>
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<center>
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  <li style="display: inline-block;"> [[File:T--TUDelft--plateL1s.jpg|thumb|none|550px|<b>Figure 1:</b> The functioning of this part has been confirmed qualitatively in <i>E. coli</i> (L1U).]] </li>
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</center>
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    </ul></div>
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===References===
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<html>
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<ul>
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<li>
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<a id="Segall2018" href="https://www.nature.com/articles/nbt.4111" target="_blank">
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Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. <i>Nature Biotechnology</i>, 36(4), 352–358.</a>
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</li>
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<li>
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<a id="Weber2011" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765" target="_blank">
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S. Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. Plos ONE, 6(2), e16765. doi: 10.1371/journal.pone.0016765 </a>
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</li>
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</ul>
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</html>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 17:44, 6 December 2019

PBHR sp1 promoter

Broad host range promoter with a binding site for TALE (Transcriptional Activator like Effector) repressor protein.

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

The broad host range promoter (PBHR) has been designed by combining promoter regions of E. coli, B. subtilis and S. cerevisiae. The promoter is based on the Pmin minimal promoter of S. cerevisiae. It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in E. coli and B. subtilis . Therefore, to make the Pmin promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the Pmin promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′.
The broad host range promoter (PBHR) was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). The 17bp spacer between the -35 and -10 conserved sequences (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) was replaced with a binding site (16bp) for TALE protein. TALE proteins consist of repeats where 12th and 13th amino acids can vary, the repeats are called the repeat variable diresidue (RVD) (Segall-Shapiro et al., 2018). These RVDs have been shown to bind to DNA in a simple one-to-one binding code. A unique 16bp binding site was incorporated into the promoter, this binding site recruits a specific TALE protein called TALEsp1 that acts as a repressor (Segall-Shapiro et al., 2018). The TALEsp1 protein was designed to bind protein specifically at the binding site (Segall-Shapiro et al., 2018).

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 pICH41233 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 (Weber et al., 2011). 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). Click here for the protocol.


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 here.


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

Characterization

  • Figure 1: The functioning of this part has been confirmed qualitatively in E. coli (L1U).

References