Difference between revisions of "Part:BBa K2918014"

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===Overview===
 
===Overview===
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This ribosomal binding site has been made by the use of the RBS calculator 2.0 by <html><a href="#Sen2017">(Yang et al., 2017)</a></html>. This RBS functions both in gram negative (<i>E. coli</i>) and gram positive (<i>B. subtilis</i>). It has been demonstrated to work in <i> P. putida </i>.
+
This ribosomal binding site has been made by the use of the RBS calculator 2.0 by <html><a href="#Yang2017">(Yang et al., 2017)</a></html>. The RBS functions both in gram negative (<i>E. coli</i>) and gram positive (<i>B. subtilis</i>). It has been demonstrated to work in <i> P. putida</i>.
  
 
===Strain Construction===
 
===Strain Construction===
−
The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible 5'UTR overhangs. For this particular RBS, the downstream overhang 'AATG' can be modified to 'ATG' since the RBS sequence ends with adenine. This design choice will allow for seamless cloning between the 5'UTR and coding sequence leaving no part-junction sequences. The RBS was then cloned in a level 0 MoClo backbone <html><a href="http://www.addgene.org/47992">pICH41246</a></html> and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning section below.
+
The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible 5' UTR overhangs. For this particular RBS, the downstream overhang 'AATG' can be modified to 'ATG' since the RBS sequence ends with adenine. This design choice will allow for seamless cloning between the 5' UTR and coding sequence leaving no part-junction sequences. The RBS was then cloned in a level 0 MoClo backbone <html><a href="http://www.addgene.org/47992">pICH41246</a></html> and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning 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).
+
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).
 
For the protocol, you can find it <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html>
 
For the protocol, you can find it <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html>
  
  
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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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</html>
 
</html>
  
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===References===
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===Characterization===
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<html>
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The Universal RBS was characterized by comparing it to <html><body><a href="https://parts.igem.org/Part:BBa_B0032"> BBa_B0032</a></body></html>. For comparision of the two RBSs, they were cloned in the same backbone <html><body><a href="http://www.addgene.org/48003/">(pICH47761)</a></body></html> and were paired with the same promoter <html><body><a href="https://parts.igem.org/Part:BBa_K2918010"> (T7<sub>sp1</sub>)</a></body></html>. The strengths were compared by measuring fluorescence readout from <html><body><a href="https://parts.igem.org/Part:BBa_K2918037"> harmonized GFP </a></body></html> by flow cytometry and <i> E. coli </i> BL21 (DE3) cells were used as blank. Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a> </html> for the protocol. FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment.
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<ul>
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<br>
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<li>
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−
<a id="Sen2017" href="https://academic.oup.com/nar/article/40/17/8773/2411560" 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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</li>
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</ul>
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</html>
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The scatter plot in figure 1 was used to gate the most dense cell regions of the blank and the same gating was considered to obtain the fluorescence values depicted in figure 2. Cells of similar forward and side scatter were compared.
  
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===Characterization===
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<div><ul>  
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  <p> The Universal RBS was characterized by comparing it to  <html><body><a href="https://parts.igem.org/Part:BBa_B0032"> BBa_B0032 </a></body></html>. As a reporter, a GFP fluorescence readout was used.  In order to measure fluorescence, we use a flow cytometer.  <br>
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<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--URBSscatter.png|thumb|none|550px|<b>Figure 1: </b>Scatter plot of forward and side scatter of <i>E. coli</i> BL21 (DE3) cells.]] </li>
 +
</center>
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    </ul></div>
  
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The GFP used as readout was our <html><body><a href="https://parts.igem.org/Part:BBa_K2918037"> harmonized eGFP </a></body></html>. All of the 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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<p>Gating was performed on the data in the fluorescnece histogram (figure 2) to discern between fluorescent and non-fluorescent cells. </p>
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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 grow 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 reference. 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 2 shows the gating based on size and figure 3 shows the fluorescence histogram of each sample. </p>
 
  
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<html><body><img src = "https://static.igem.org/mediawiki/parts/a/a0/T--TUDelft--URBS.svg" alt="Modeling" style="width:60%";></body></html>
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<div><ul>  
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<html><body><figcaption><br><b>Figure 1: Fluorescence values of T7, T7sp1 with median fluorescence of <i>E. coli BL21</i> cells without a plasmid substracted. </b></figcaption></body></html>
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<center>
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  <li style="display: inline-block;"> [[File:T--TUDelft--URBSfluorescence.png|thumb|none|550px|<b>Figure 2: Raw fluorescence data. The curves represent fluorscenece values of <i>E. coli</i> BL21 (DE3) cells (black), clones with BBa_B0032 (red) and clones with Universal RBS (blue).</b>]] </li>
 +
</center>
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    </ul></div>
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<br>
 
<br>
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<p> From figure 2 the median fluorescence intensity of the two samples was obatined and corrected by the fluorescence of <i>E. coli</i> BL21 (DE3) cells. Figure 3 depicts the fluorescence of GFP expression influenced by BBa_B0032 and Universal RBS.
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</p>
  
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<p>Figure 1 shows that the strength of our Universal RBS in <i>E. coli</i> is about 60% higher than <html><body><a href="https://parts.igem.org/Part:BBa_B0032"> BBa_B0032 </a></body></html>.
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<div><ul>
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<br> <br>
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<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--URBS.svg|thumb|none|550px|<b>Figure 3: Fluorescence values of for BBa_B0032 and Universal RBS with median fluorescence of <i>E. coli BL21</i> cells without a plasmid substracted. </b>]] </li>
 +
</center>
 +
    </ul></div>
  
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<html><body><img src = "https://static.igem.org/mediawiki/parts/b/be/T--TUDelft--URBSscatter.png" alt="Modeling" style="width:60%";></body></html>
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From figure 3, strength of universal RBS is shown to be 60% higher than BBa_B0032.
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<html><body><figcaption><br><b>Figure 2: Scatter plot of forward and side scatter of <i>E. coli Top 10</i> cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 1. </b></figcaption></body></html>
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<br>
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<html><body><img src = "https://static.igem.org/mediawiki/parts/4/46/T--TUDelft--URBSfluorescence.png" alt="Modeling" style="width:60%";></body></html>
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===References===
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<html><body><figcaption><br><b>Figure 3: Raw fluorescence values. Black is <i>E. coli Top 10</i> cells without a plasmid. Red is T7 and blue is T7<sub>sp1<sub> </b></figcaption></body></html>
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<html>
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<br>
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<ul>
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 +
<li>
 +
<a id="Weber2011" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765" target="_blank">
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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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<li>
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                        <a id="Yang2017" href="https://www.ncbi.nlm.nih.gov/pubmed/29061047" target="_blank">
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                            Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." <u> ACS Synthetic Biology </u> 7(1): 287-291.
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                        </a>
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                    </li>
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</ul>
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</html>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2918014 parameters</partinfo>
 
<partinfo>BBa_K2918014 parameters</partinfo>
 
<!-- -->
 
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Latest revision as of 18:00, 6 December 2019

Universal RBS

Ribosome Binding Site which is functional in both gram positive and gram negative bacteria.

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]

The part has been confirmed by sequencing and has no mutations.

Overview

This ribosomal binding site has been made by the use of the RBS calculator 2.0 by (Yang et al., 2017). The RBS functions both in gram negative (E. coli) and gram positive (B. subtilis). It has been demonstrated to work in P. putida.

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible 5' UTR overhangs. For this particular RBS, the downstream overhang 'AATG' can be modified to 'ATG' since the RBS sequence ends with adenine. This design choice will allow for seamless cloning between the 5' UTR and coding sequence leaving no part-junction sequences. The RBS was then cloned in a level 0 MoClo backbone pICH41246 and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning 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). 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

Characterization

The Universal RBS was characterized by comparing it to BBa_B0032. For comparision of the two RBSs, they were cloned in the same backbone (pICH47761) and were paired with the same promoter (T7sp1). The strengths were compared by measuring fluorescence readout from harmonized GFP by flow cytometry and E. coli BL21 (DE3) cells were used as blank. Click here for the protocol. FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment.

The scatter plot in figure 1 was used to gate the most dense cell regions of the blank and the same gating was considered to obtain the fluorescence values depicted in figure 2. Cells of similar forward and side scatter were compared.

  • Figure 1: Scatter plot of forward and side scatter of E. coli BL21 (DE3) cells.


Gating was performed on the data in the fluorescnece histogram (figure 2) to discern between fluorescent and non-fluorescent cells.


  • Figure 2: Raw fluorescence data. The curves represent fluorscenece values of E. coli BL21 (DE3) cells (black), clones with BBa_B0032 (red) and clones with Universal RBS (blue).


From figure 2 the median fluorescence intensity of the two samples was obatined and corrected by the fluorescence of E. coli BL21 (DE3) cells. Figure 3 depicts the fluorescence of GFP expression influenced by BBa_B0032 and Universal RBS.

  • Error creating thumbnail: File missing
    Figure 3: Fluorescence values of for BBa_B0032 and Universal RBS with median fluorescence of E. coli BL21 cells without a plasmid substracted.

From figure 3, strength of universal RBS is shown to be 60% higher than BBa_B0032.

References