View source for Part:BBa K2918014

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<partinfo>BBa_K2918014 short</partinfo>

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

<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2918014 SequenceAndFeatures</partinfo>

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 <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===
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 (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>


<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>Table 1:</b> Overview of different level in MoClo
        <table id="tabletu">
            <tr>
                <th>Level
                </th>
                <th>Basic/Composite
                </th>
                <th>
                    Type</th>
                <th>Enzyme</th>
            </tr>
            <tr>
                <td>
                    Level 0
                </td>
                <td>Basic</td>
                <td>Promoters, 5’ UTR, CDS and terminators</td>
                <td>BpiI</td>

            </tr>
            <tr>        <td>Level 1</td>
                <td>Composite</td>
                <td>Transcriptional units</td>
                <td>BsaI</td>
            </tr>
            <tr>
                <td>
                    Level 2/M/P</td>
                <td>Composite</td>
                <td>Multiple transcriptional units</td>
                <td>BpiI</td>
            </tr>


        </table>


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For synthesizing basic parts, the part of interest should be flanked by a <span style="color:limegreen">BpiI site</span> and its <span style="color:dodgerblue">specific type overhang</span>. 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.



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        <b>Table 2:</b> Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts
        <table id="tabletu">
            <tr>
                <th>Basic Part
                </th>
                <th>Sequence 5' End
                </th>
                <th>
                    Sequence 3' End</th>
                <th>Level 0 backbone</th>
            </tr>
            <tr>
                <td>
                    Promoter
                </td>
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GGAG</span></td>
                <td><span style="color:dodgerblue">TACT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
                <td>pICH41233</td>

            </tr>
            <tr>        <td>5’ UTR</td>
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">TACT</span></td>
                <td><span style="color:dodgerblue">AATG</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
                <td>pICH41246</td>
            </tr>
            <tr>
                <td>
                    CDS</td>
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">AATG</span></td>
                <td><span style="color:dodgerblue">GCTT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
                <td>pICH41308</td>
            </tr>
            <tr>
                <td>
                    Terminator</td>
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GCTT</span></td>
                <td><span style="color:dodgerblue">CGCT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
                <td>pICH41276</td>
            </tr>


        </table>


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===Characterization===
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 DE(3) 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.
<br>

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.

<div><ul> 
<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 DE(3) cells.]] </li>
</center>
    </ul></div>


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


<div><ul> 
<center>
   <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 DE (3) cells (black), clones with BBa_B0032 (red) and clones with Universal RBS (blue).</b>]] </li>
</center>
    </ul></div>

<br>
<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 DE(3) cells. Figure 3 depicts the fluorescence of GFP expression influenced by BBa_B0032 and Universal RBS. 
</p>

<div><ul> 
<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>

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

===References===
<html>
<ul>

<li>
<a id="Weber2011" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765" target="_blank">
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>
</li>
<li>
                        <a id="Yang2017" href="https://www.ncbi.nlm.nih.gov/pubmed/29061047" target="_blank">
                            Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." <u> ACS Synthetic Biology </u> 7(1): 287-291.
                        </a>
                    </li>
</ul>

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===Functional Parameters===
<partinfo>BBa_K2918014 parameters</partinfo>
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