Difference between revisions of "Part:BBa K2918009"
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | The <html> <a | + | The <html> <a href="https://parts.igem.org/Part:BBa_K2918006">medium T7 promoter variant</a> </html> was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). 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 18bp binding site was incorporated into the promoter, this binding site recruits a specific TALE protein called <html> <a href="https://parts.igem.org/Part:BBa_K2918008">TALEsp1</a> </html> 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 and are predicted not to bind anywhere in the <i> E. coli </i> genome. The position of the binding site can be readjusted to alter the extent of repression by the TALE protein. |
+ | <div><ul> | ||
+ | <center> | ||
+ | <li style="display: inline-block;"> [[File:T--TUDelft--T7.png|thumb|none|550px|<b>Figure 1: Design of TALEsp1-regulated version of T7 promoter variants was done by annexing the operator sequence directly downstream of the 23 base pair promoter. </b>]] </li> | ||
+ | </center> | ||
+ | </ul></div> | ||
===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. | + | <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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</body> | </body> | ||
</html> | </html> | ||
− | |||
===Characterization=== | ===Characterization=== | ||
− | <p>The strength of the medium T7<sub>sp1</sub> promoter was characterized by comparing it to <html><a href="https://parts.igem.org/Part:BBa_K2918010"> T7<sub>sp1<sub> promoter</a></body></html>. For comparision of the promoter strengths, the two promoters were cloned in the same backbone <html><body><a href="http://www.addgene.org/47761/">(pICH47761)</a></body></html> and were paired with the same RBS <html><body><a href="https://parts.igem.org/Part:BBa_K2918014"> (Universal RBS)</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 | + | <p>The strength of the medium T7<sub>sp1</sub> promoter was characterized by comparing it to <html><a href="https://parts.igem.org/Part:BBa_K2918010"> T7<sub>sp1</sub> promoter</a></body></html>. For comparision of the promoter strengths, the two promoters were cloned in the same backbone <html><body><a href="http://www.addgene.org/47761/">(pICH47761)</a></body></html> and were paired with the same RBS <html><body><a href="https://parts.igem.org/Part:BBa_K2918014"> (Universal RBS)</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. FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment. Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a> </html> for the protocol. |
<br> | <br> | ||
− | The scatter plot in figure | + | The scatter plot in figure 2 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 3. Cells of similar forward and side scatter were compared.</p> |
<div><ul> | <div><ul> | ||
<center> | <center> | ||
− | <li style="display: inline-block;"> [[File:T--TUDelft--0.5T7sp1charsscatter.png|thumb|none|550px|<b>Figure | + | <li style="display: inline-block;"> [[File:T--TUDelft--0.5T7sp1charsscatter.png|thumb|none|550px|<b>Figure 2: Scatter plot of forward and side scatter of <i>E. coli</i> BL21 (DE3) cells. </b>]] </li> |
</center> | </center> | ||
</ul></div> | </ul></div> | ||
− | <p>Gating was performed on the data in the fluorescence histogram (figure | + | <p>Gating was performed on the data in the fluorescence histogram (figure 3) to discern between fluorescent and non-fluorescent cells. </p> |
<div><ul> | <div><ul> | ||
<center> | <center> | ||
− | <li style="display: inline-block;"> [[File:T--TUDelft--0.5T7sp1charsfluorescence.png|thumb|none|550px|<b>Figure | + | <li style="display: inline-block;"> [[File:T--TUDelft--0.5T7sp1charsfluorescence.png|thumb|none|550px|<b>Figure 3 Raw fluorescence data. The curves represent fluorscenece values of <i>E. coli</i> BL21 (DE3) cells (black), clones with GFP expressed from T7<sub>sp1</sub> (red) and clones with GFP expressed from T7<sub>sp1</sub> (blue).</b>]] </li> |
</center> | </center> | ||
</ul></div> | </ul></div> | ||
− | <p> From figure | + | <p> From figure 3 the median fluorescence intensity of the two samples was obtained and corrected by the fluorescence of <i>E. coli</i> BL21 (DE3). Figure 4 depicts the fluorescence of GFP expression controlled by T7<sub>sp1</sub> and medium T7<sub>sp1</sub>. |
</p> | </p> | ||
<div><ul> | <div><ul> | ||
<center> | <center> | ||
− | <li style="display: inline-block;"> [[File:T--TUDelft--T7characterisation.png|thumb|none|550px|<b>Figure | + | <li style="display: inline-block;"> [[File:T--TUDelft--T7characterisation.png|thumb|none|550px|<b>Figure 4: Fluorescence values of medium T7sp1 and T7sp1. For correction of background fluorescence, <i>E. coli </i> BL21 (DE3) cells without a plasmid were used. </b>]] </li> |
</center> | </center> | ||
</ul></div> | </ul></div> | ||
− | <p>Figure | + | <p>Figure 4 shows that the strength of medium T7<sub>sp1</sub> is approximately 64% relative to T7<sub>sp1</sub> promoter. We can thus conclude that the binding site for TALEsp1 does not influence the strength of the promoter as the strength of medium T7 promoter has been shown to be 50% of wild type T7 promoter <html><a href="#Komura2018">(Komura et al., 2018)</a></html> |
<br> <br> | <br> <br> | ||
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<li> | <li> | ||
<a id="Weber2011" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765" target="_blank"> | <a id="Weber2011" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765" target="_blank"> | ||
− | + | 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="Segall2018" href="https://www.nature.com/articles/nbt.4111" target="_blank"> | <a id="Segall2018" href="https://www.nature.com/articles/nbt.4111" target="_blank"> |
Latest revision as of 17:46, 6 December 2019
Medium T7sp1 promoter
T7 promoter variant with about 50% strength relative to wild type T7 promoter and has been engineered to contain a binding site for TALE repressor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
This part has been confirmed by sequencing and has no mutations.
Usage and Biology
The medium T7 promoter variant was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). 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 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 et al., 2018). The TALEsp1 protein was designed to bind protein specifically at the binding site and are predicted not to bind anywhere in the E. coli genome. The position of the binding site can be readjusted to alter the extent of repression by the TALE protein.
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.
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 strength of the medium T7sp1 promoter was characterized by comparing it to T7sp1 promoter