Difference between revisions of "Part:BBa K2918000"

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===Usage and Biology===
 
===Usage and Biology===
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The broad host range promoter 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 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′.
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The broad host range promoter 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′.
  
 
===Strain Construction===
 
===Strain Construction===
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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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         </table>
 
         </table>
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     </body>
 
     </body>
 
</html>
 
</html>
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===Characterization===
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  <p> The P<sub>Bhr</sub> was characterized by comparing it to a T7 promoter. As a reporter, a GFP fluorescence readout was used.  In order to measure fluorescence, we use a flow cytometer.  <br>
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The GFP used as readout was Juniper GFP <html><body><a href="https://parts.igem.org/Part:BBa_J97001">BBa_J97001</a></body></html>. The ribosome binding site was our <html><body><a href="https://parts.igem.org/Part:BBa_K2918014">Universal RBS</a></body></html>. All of these parts were cloned into a level 1 backbone <html><body><a href="http://www.addgene.org/47761/">pICH47761</a></body></html>.
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<br>
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The protocol for preparation of samples for the flow cytometry assay is as follows:
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<html>
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<body>
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<ol>
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<li>Samples were grown overnight</li>
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<li>Overnight cultures were diluted to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes. </li>
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<li>Overnight cultures were diluted 1:100 into 5 mL, and grown for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with 1 mM IPTG where necessary </li>
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<li>Samples were kept at 4 degrees for 1 hour </li>
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</ol>
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</body>
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</html>
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<br>
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In the measurement, <i>E. coli BL21</i> cells without a plasmid were used as a blank. The gating for flow cytometry was determined by eye by selecting the densest region of the blank. Furthermore, the fluorescence histogram was gated to discern between cells that were 'on' and 'off', as in expressing fluorescence or not. Only cells of similar forward and side scatter were compared.  The median fluorescence intensity of the blank is subtracted from the fluorescence intensity of the samples to correct for autofluorescence. In figure 1 we plot the corrected fluorescence of the samples. </p>
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<div><ul>
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<center>
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  <li style="display: inline-block;"> [[File:T--TUDelft--Pbhrcharacterisation.png|thumb|none|550px|<b>Figure 1:</b> Fluorescence corrected for autofluorescence of  <i>E. coli BL21</i> cells without a plasmid. ]] </li>
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</center>
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    </ul></div>
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<p>Figure 1 shows that the strength of P<sub>Bhr</sub> in <i>E. coli</i> is significantly higher than a T7 promoter induced using 1 mM IPTG for 4 hours. P<sub>Bhrsp1 v2</sub> contains the binding site for TALEsp1. The presence of the binding site seems to reduce the strength of the promoter. </p>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 16:58, 6 December 2019

PBHR

Broad host range promoter demonstarted to work in E.coli and P. putida and expected to work in B. subtilis

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 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′.

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. 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.


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

The PBhr was characterized by comparing it to a T7 promoter. As a reporter, a GFP fluorescence readout was used. In order to measure fluorescence, we use a flow cytometer.
The GFP used as readout was Juniper GFP BBa_J97001. The ribosome binding site was our Universal RBS. All of these parts were cloned into a level 1 backbone pICH47761.
The protocol for preparation of samples for the flow cytometry assay is as follows:

  1. Samples were grown overnight
  2. Overnight cultures were diluted to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes.
  3. Overnight cultures were diluted 1:100 into 5 mL, and grown for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with 1 mM IPTG where necessary
  4. Samples were kept at 4 degrees for 1 hour

In the measurement, E. coli BL21 cells without a plasmid were used as a blank. The gating for flow cytometry was determined by eye by selecting the densest region of the blank. Furthermore, the fluorescence histogram was gated to discern between cells that were 'on' and 'off', as in expressing fluorescence or not. Only cells of similar forward and side scatter were compared. The median fluorescence intensity of the blank is subtracted from the fluorescence intensity of the samples to correct for autofluorescence. In figure 1 we plot the corrected fluorescence of the samples.


  • Figure 1: Fluorescence corrected for autofluorescence of E. coli BL21 cells without a plasmid.


Figure 1 shows that the strength of PBhr in E. coli is significantly higher than a T7 promoter induced using 1 mM IPTG for 4 hours. PBhrsp1 v2 contains the binding site for TALEsp1. The presence of the binding site seems to reduce the strength of the promoter.