Difference between revisions of "Part:BBa K3758000"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3758000 short</partinfo>
 
<partinfo>BBa_K3758000 short</partinfo>
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__TOC__
  
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<html>
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<style>
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.mw-body a.external, .link-https { padding-right: 0 }
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#bodyContent a[href^="https://"], .link-https { padding-right: 0 }
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===Usage and Biology===
 
  
−
<h2>Overview</h2>
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 +
==Part Description==
 
<p>
 
<p>
−
This part was created using the Phytobrick Entry Vector with GFP dropout <a href="https://2021.igem.org/Team:Marburg/Parts">Basic Parts</a><a href="https://parts.igem.org/Part:BBa_K2560002">BBa_K2560002</a>(https://parts.igem.org/Part:BBa_K2560002) and was designed to be compatible with the Phytobrick assembly standard. All parts created this year were acquired via PCR from purified DNA samples using a CTAB based method (https://doi.org/10.1186/s42269-019-0066-1), by primer annealing, primer annealing, and extension reactions or synthesized via IDT/Twist.</p><br>
+
This part was created using the Phytobrick Entry Vector with GFP dropout [https://parts.igem.org/Part:BBa_K2560002 BBa_K2560002] and was designed to be compatible with the Phytobrick assembly standard. This years BioBricks were either constructed via PCR from purified DNA samples using a CTAB based method <sup>[https://doi.org/10.1186/s42269-019-0066-1 <nowiki>[1]</nowiki>]</sup>, by primer annealing, primer annealing, and extension reactions or synthesized via IDT/Twist.</p>
  
  
 
<p>
 
<p>
−
All parts this year were produced to be used in the chloroplast of different plant species. For the characterization of these parts they were tested in chloroplast cell-free systems (ccfs) from either the same species or they were tested in ccfs from other plant species. Plastid parts offer the benefit of highly conserved regulatory sequences that can be used across species. Although characterizing chloroplast parts is a huge effort, in literature, it has been shown that plastid parts can be used across species to drive gene expression (https://doi.org/10.7554/eLife.13664.008). We believe that based on this knowledge we can create valuable parts that can be screened for activity in our system with the final goal of building a variety of different parts. This collection shall help combat unwanted recombination events in vivo that sometimes impede the successful functionality of the genetic design.
+
All parts this year were produced to be used in the chloroplast of different plant species. For the characterization of these parts they were tested in chloroplast cell-free systems (ccfs) from either the same species or they were tested in ccfs from other plant species. Plastid parts offer the benefit of highly conserved regulatory sequences that can be used across species. Although characterizing chloroplast parts is a huge effort, in literature, it has been shown that plastid parts can be used across species to drive gene expression <sup>[https://doi.org/10.7554/eLife.13664 <nowiki>[2]</nowiki>]</sup>. We believe that based on this knowledge we can create valuable parts that can be screened for activity in our system with the final goal of building a variety of different parts. This collection shall help combat unwanted recombination events in vivo that sometimes impede the successful functionality of the genetic design.
 
</p>
 
</p>
  
 
<h3>The PEP Promoter</h3>
 
<h3>The PEP Promoter</h3>
−
<p>Transcription in the chloroplast is mainly driven by two different RNA Polymerases: plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP).<br>
+
<p>Transcription in the chloroplast is mainly driven by two different RNA Polymerases: plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP). <br>
−
The PEP is a bacterial-like polymerase that is a remnant of the chloroplasts cyanobacterial ancestor and is only capable of promoting gene expression in the plastid. These polymerases are able to interact with nuclear-encoded sigma factors and therefore are able to recognize bacterial promoter motives such as the -35 (TTGACA) and the Pribnow (TATAAT) box. Similarly to bacteria there are different sigma factors promoting gene expression under different growth conditions. As the PEP is structurally more sophisticated there are even more peptides involved in DNA transcription that are not fully understood yet. </p><br>
+
The PEP is a bacterial-like polymerase that is a remnant of the chloroplast’s cyanobacterial ancestor and is only capable of promoting gene expression in the plastid. These polymerases are able to interact with nuclear-encoded sigma factors and therefore are able to recognize bacterial promoter motives such as the -35 (TTGACA) and the Pribnow (TATAAT) box. Similar to bacteria there are different sigma factors promoting gene expression under different growth conditions. As the PEP is structurally more sophisticated there are even more peptides involved in DNA transcription that is not fully understood yet.
 +
</p><br>
 
<h3>The NEP Promoter</h3>
 
<h3>The NEP Promoter</h3>
−
The NEP is a T3/T7 phage-like polymerase that is encoded in the nucleus and is imported into the chloroplast. It was proposed that this polymerase is a remnant of a horizontal gene transfer from a bacterium to a eubacterial ancestor of today's plant cells [1][2].This type of polymerase mainly promotes gene expression in early developmental stages of the chloroplast. In mature chloroplasts it continues to transcribe housekeeping genes like the subunits of the plastid encoded polymerase (rpoA, rpoB, rpoC1 and rpoC2) and proteins involved in fatty acid biosynthesis such as acetyl-CoA carboxylase (accD). In contrast the PEP is rather active in mature chloroplasts and is primarily involved in the expression of photosynthetic genes. For other non-photosynthetic genes, motives of both polymerases can be found and it has been shown that both can promote transcription using deletion studies of important promoter sequences. (Hier Link zu einen von Maligas Papern).
+
<p>
 +
The NEP is a T3/T7 phage-like polymerase that is encoded in the nucleus and is imported into the chloroplast. It was proposed that this polymerase is a remnant of a horizontal gene transfer from a bacterium to a eubacterial ancestor of today's plant cells <sup>[https://doi.org/10.1016/j.tim.2005.08.012 <nowiki>[3]</nowiki>]</sup> <sup>[https://doi.org/10.1007/4735_2007_0232 <nowiki>[4]</nowiki>]</sup>. This type of polymerase mainly promotes gene expression in the early developmental stages of the chloroplast. In mature chloroplasts, it continues to transcribe housekeeping genes like the subunits of the plastid-encoded polymerase (rpoA, rpoB, rpoC1, and rpoC2) and proteins involved in fatty acid biosynthesis such as acetyl-CoA carboxylase (accD). In contrast, the PEP is rather active in mature chloroplasts and is primarily involved in the expression of photosynthetic genes. For other non-photosynthetic genes, motives of both polymerases can be found and it has been shown that both can promote transcription using deletion studies of important promoter sequences <sup>[https://doi.org/10.1007/s00294-002-0293-z <nowiki>[5]</nowiki>]</sup> <sup>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC166747/ <nowiki>[6]</nowiki>]</sup>.
 +
</p>
 +
 
 +
==Characterization & Measurement==
 +
 
 +
<h3>Batch effect</h3>
 +
 
 +
<p>
 +
A major difficulty when working with cell free technology is the reproducibility of reliable data generation. While one batch of cell free extract generation might be perfectly suited for efficient protein production, other batches might not perform that well. Because our chloroplast extracts were prepared from leaves, each preparation could have contained distinct compositions of differentiated leaf cells (for example:
 +
mesophyll, palisade parenchyma and bundle sheath) from plants that experienced microclimatic variations. This in turns causes the individual difference in expression strength, making it impossible to quantitatively compare data across measurements.
 +
 
 +
 
 +
</p>
 +
 
 +
<h3>Dual Luciferase Normalization</h3>
 +
 
 +
<p>
 +
 
 +
To tackle the aforementioned issue, we designed our measurement construct to harbor two individual luciferase cassettes. A standardized cassette that includes the Firefly luciferase (FLuc) is included in every measurement construct in order to have a reference point to which we can normalize our gained data to<sup>[https://doi.org/10.1038/nmeth.3659 <nowiki>[14]</nowiki>] </sup>.
 +
The second cassette consists of a Nano Luciferase (NLuc) harboring a placeholder part in either the promoter, 5’UTR or 3’UTR position. This enables quick exchange of a series of lvl0 parts allowing for high throughput characterization of genetic parts.
 +
 
 +
</p>
 +
 
 +
<html>
 +
<center>
 +
<figure>
 +
    <img src='https://static.igem.org/mediawiki/parts/3/31/T--Marburg--Dual_Luciferase_SBOL.svg' width='600px' alt='sfGFP_Cell-Free_Tobacco' />
 +
    <figcaption><p><b>Figure 1: SBOL Visualization of our Dual Luciferase lvl2 measurement vector</b><br>
 +
On the left you can see the inverted Firefly luciferase that is used for ratiometric normalization of our data.<br>
 +
The right cassette shows the NanoLuc luciferase, in which Dropout sequences have been included for rapid exchange of parts in either the promoter, 5'UTR or 3'UTR position</figcaption>
 +
</figure></center></html>
 +
 
 +
 
 +
<h3>Placeholder</h3>
 +
 
 +
<p>
 +
For the characterization of the parts produced this year, we made use of Golden Gate placeholder parts introduced by [http://2019.igem.org/Team:Marburg iGEM Marburg 2019]:
 +
[https://parts.igem.org/Part:BBa_K3228060 BBa_K3228060],
 +
[https://parts.igem.org/Part:BBa_K3228061 BBa_K3228061] and
 +
[https://parts.igem.org/Part:BBa_K3228063 BBa_K3228063]. These placeholder parts can be used in assemblies to subsequently replace it with another part of the same type in a secondary Golden Gate assembly. A placeholder can rationalize large-scale assemblies: Instead of building each plasmid from scratch, a placeholder is used to generate an entry vector.
 +
 
 +
</p>
 +
 
 +
==Results==
 +
 
 +
 
 +
==Marburg collection 3.0==
 +
 
 +
<p>
 +
We proudly present the third expansion of the Marburg collection <sup>[https://doi.org/10.1021/acssynbio.1c00126 <nowiki>[15]</nowiki>]</sup>. The Marburg collection is a Golden Gate based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. The collection overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids.
 +
36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units.<br><br>
 +
The original [http://2018.igem.org/Team:Marburg/Part_Collection Marburg Collection] contains 123 parts in total, including:
 +
inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. The toolbox was constructed as a foundation for future iGEM teams to empower accelerated progression in their ambitious projects.
 +
<br><br>
 +
Our collection includes genetic parts suitable for use in the chloroplast. Among parts from the chloroplast of <i>Nicotiana tabacum</i>, we built parts from the chloroplast of <i>Spinacia oleracea</i>, <i>Oryza sativa</i>,<i>Triticum aestivum</i> and <i>Quercus robur</i>. With our contribution, we aim to accelerate research in the field of plastid engineering.
 +
</p>
 +
 
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3758000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3758000 SequenceAndFeatures</partinfo>
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===References===
 
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[1] Filée, J., & Forterre, P. (2005). Viral proteins functioning in organelles: a cryptic origin? Trends in Microbiology, 13(11), 510–513. https://doi.org/10.1016/j.tim.2005.08.012
 
  
−
[2] Liere, K., & Börner, T. (2007). Transcription and transcriptional regulation in plastids. In Cell and Molecular Biology of Plastids (pp. 121–174). Springer Berlin Heidelberg. https://doi.org/10.1007/4735_2007_0232
 
  
−
[3] Suzuki, J. Y., Sriraman, P., Svab, Z., & Maliga, P. (2003). Unique Architecture of the Plastid Ribosomal RNA Operon Promoter Recognized by the Multisubunit RNA Polymerase in Tobacco and Other Higher Plants. The Plant Cell, 15(1), 195–205. https://doi.org/10.1105/tpc.007914
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==Overview Marburg collection 3.0==
 +
 
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<div class="PCListIcon" style="display:flex;flex-direction:row; flex-wrap: nowrap; justify-content:space-evenly; align-items:flex-start;font-size:100%;">
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[[File:T--Marburg--5'Homology.png|120px|thumb|none|
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<html>
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<ul>
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<b>
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<li> <a href="https://parts.igem.org/Part:BBa_K3758250">K3758250 </a><br> trnG 5'Homology Nt </li>
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<li> <a href="https://parts.igem.org/Part:BBa_K3758252">K3758252 </a><br> trnI 5'Homology Nt </li>
 +
 
 +
</b>
 +
</ul>
 +
</html>]]
 +
 
 +
[[File:T--Marburg--Promoter_Symbol.png|120px|thumb|none|
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 +
<html>
 +
<ul>
 +
<b>
 +
 
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758000">K3758000 </a><br> Prrn (-64 to +17) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758001">K3758001 </a><br> Prrn (5fold decrease) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758002">K3758002 </a><br> PpsbA Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758003">K3758003 </a><br> PpsbA (-42 to +9) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758004">K3758004 </a><br> PpsbB Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758005">K3758005 </a><br> PpsbD-leader Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758006">K3758006 </a><br> PaccD (-84 to +23) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758007">K3758007 </a><br> PaccD (functional) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758008">K3758008 </a><br> PatpB (+NEP-PEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758009">K3758009 </a><br> PatpB (only NEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758010">K3758010 </a><br> PatpB (+NEP+PEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758011">K3758011 </a><br> PatpB (only PEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758012">K3758012 </a><br> PatpH Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758013">K3758013 </a><br> PatpI (PEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758014">K3758014 </a><br> PclpP (NEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758015">K3758015 </a><br> PclpP (PEP) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758016">K3758016 </a><br> PrbcL Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758017">K3758017 </a><br> PrbcL (core -35 to +9) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758018">K3758018 </a><br> PrpoB Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758019">K3758019 </a><br> Prrn1 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758020">K3758020 </a><br> Prrn2 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758021">K3758021 </a><br> Prrn3 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758022">K3758022 </a><br> Prrn5 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758023">K3758023 </a><br> Prrn6 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758024">K3758024 </a><br> Prrn7 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758025">K3758025 </a><br> Prrn8 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758026">K3758026 </a><br> Prrn9 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758027">K3758027 </a><br> Prrn10 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758028">K3758028 </a><br> Prrn12 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758029">K3758029 </a><br> Prrn13 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758030">K3758030 </a><br> Prrn14 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758031">K3758031 </a><br> Prrn15 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758032">K3758032 </a><br> Prrn16 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758033">K3758033 </a><br> Prrn17 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758034">K3758034 </a><br> Prrn18 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758035">K3758035 </a><br> Prrn19 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758036">K3758036 </a><br> Prrn20 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758037">K3758037 </a><br> Prrn21 (library) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758038">K3758038 </a><br> PpsbD Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758039">K3758039 </a><br> PpsbK Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758040">K3758040 </a><br> PpsbA Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758041">K3758041 </a><br> PrbcL Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758042">K3758042 </a><br> Prrn16 (short) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758043">K3758043 </a><br> Prrn16 (long) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758044">K3758044 </a><br> Prrn16 Ta </li>
 +
 
 +
</ul>
 +
</b>
 +
</html>]]
 +
 
 +
[[File:T--Marburg--5'UTR_Symbol.png|120px|thumb|none|
 +
 
 +
<html>
 +
<ul>
 +
<b>
 +
    <li> <a href="https://parts.igem.org/Part:BBa_K3758070">K3758070 </a><br> atpB 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758071">K3758071 </a><br> atpB 5'UTR (+10) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758072">K3758072 </a><br> atpB 5'UTR (-90 to +10 processed) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758073">K3758073 </a><br> atpB 5'UTR (TCR processed -90) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758074">K3758074 </a><br> atpH 5'UTR (-45 processed) 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758075">K3758075 </a><br> psbN 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758076">K3758076 </a><br> psbN 5'UTR (processed) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758077">K3758077 </a><br> psbB 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758078">K3758078 </a><br> ndhD 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758079">K3758079 </a><br> rps2 mAAGA 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758080">K3758080 </a><br> rps2 mCCUC 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758081">K3758081 </a><br> rps2 mAAGA 5'UTR (Δ -20 to -10) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758082">K3758082 </a><br> rps2 mCCUC 5'UTR (Δ -20 to -10) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758083">K3758083 </a><br> psbH 5'UTR (Startcodon + 1AA) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758084">K3758084 </a><br> psbA 5'UTR (SNM) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758085">K3758085 </a><br> psbA 5'UTR (SEM) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758086">K3758086 </a><br> psbA 5'UTR (ΔSL) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758087">K3758087 </a><br> psbA 5'UTR (Δ5'SL) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758088">K3758088 </a><br> psbA-rbcL 5'UTR (R'SLWT) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758089">K3758089 </a><br> rps14 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758090">K3758090 </a><br> rbcL 5'UTR (full) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758091">K3758091 </a><br> rbcL 5'UTR (processed) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758092">K3758092 </a><br> rbcL 5'UTR (+14AA) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758093">K3758093 </a><br> rrn16 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758094">K3758094 </a><br> rrn16 5'UTR (+37) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758095">K3758095 </a><br> psbA 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758096">K3758096 </a><br> Synthetic RBS Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758097">K3758097 </a><br> psbC 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758098">K3758098 </a><br> accD 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758099">K3758099 </a><br> clpP 5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758100">K3758100 </a><br> rbcL 5'UTR So </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758101">K3758101 </a><br> ndhC 5'UTR So </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758102">K3758102 </a><br> ndhB 5'UTR So </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758103">K3758103 </a><br> atpB 5'UTR So </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758104">K3758104 </a><br> psaC 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758105">K3758105 </a><br> psbA 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758106">K3758106 </a><br> psbB 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758107">K3758107 </a><br> rpl23 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758108">K3758108 </a><br> rpl32 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758109">K3758109 </a><br> rps12 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758110">K3758110 </a><br> rps19 5'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758111">K3758111 </a><br> Gene10 5'UTR T7 </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758113">K3758113 </a><br> rpl33 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758114">K3758114 </a><br> rps4 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758115">K3758115 </a><br> rps12 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758116">K3758116 </a><br> rps19 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758117">K3758117 </a><br> rbcL 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758118">K3758118 </a><br> psbA 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758119">K3758119 </a><br> atpB 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758120">K3758120 </a><br> psaC 5'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758121">K3758121 </a><br> atpB 5'UTR Qr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758122">K3758122 </a><br> psaC 5'UTR Qr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758123">K3758123 </a><br> psaC 5'UTR Qr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758124">K3758124 </a><br> Synthetic 5'UTR 1 </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758125">K3758125 </a><br> Synthetic 5'UTR 2 </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758126">K3758126 </a><br> Synthetic 5'UTR 3 </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758127">K3758127 </a><br> Synthetic 5'UTR 4 </li>
 +
 
 +
   
 +
 
 +
</b>
 +
</ul>
 +
</html>]]
 +
 
 +
[[File:T--Marburg--Promoter+5'UTR_Symbol.png|120px|thumb|none|
 +
 
 +
<html>
 +
<ul>
 +
<b>
 +
    <li> <a href="https://parts.igem.org/Part:BBa_K3758140">K3758140 </a><br> PaccD+5'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758141">K3758141 </a><br> Prrn+RBS(3.9%) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758142">K3758142 </a><br> Prrn+RBS(46%) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758143">K3758143 </a><br> Prrn+RBS(60%) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758144">K3758144 </a><br> Prrn+RBS(61%) Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758145">K3758145 </a><br> Prrn+RBS(100%) Nt </li>
 +
   
 +
 
 +
</b>
 +
</ul>
 +
</html>]]
 +
 
 +
 
 +
[[File:T--Marburg--Coding_Sequence_Symbol.png|120px|thumb|none|
 +
 
 +
<html>
 +
<ul>
 +
<b>
 +
 
 +
    <li> <a href="https://parts.igem.org/Part:BBa_K3758170">K3758170 </a><br> mEmerald Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758171">K3758171 </a><br> pporRFP Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758172">K3758172 </a><br> aadA Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758173">K3758173 </a><br> FLARE-16S-aadA-GFP Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758174">K3758174 </a><br> NanoLuc Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758175">K3758175 </a><br> Firefly Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758176">K3758176 </a><br> mScarlet Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758177">K3758177 </a><br> sfGFP Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758178">K3758178 </a><br> NanoLuc Cr </li>
 +
   
 +
 
 +
</ul>
 +
</b>
 +
</html>]]
 +
 
 +
[[File:T--Marburg--3'UTR_Symbol.png|120px|thumb|none|
 +
 
 +
<html>
 +
<ul>
 +
<b>
 +
    <li> <a href="https://parts.igem.org/Part:BBa_K3758200">K3758200 </a><br> psbA 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758201">K3758201 </a><br> rbcL 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758202">K3758202 </a><br> rpoA 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758203">K3758203 </a><br> petD 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758204">K3758204 </a><br> rps4 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758205">K3758205 </a><br> petN 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758206">K3758206 </a><br> petB 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758207">K3758207 </a><br> ndhB 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758208">K3758208 </a><br> atpE 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758209">K3758209 </a><br> accD 3'UTR Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758210">K3758210 </a><br> atpA 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758211">K3758211 </a><br> ndhJ 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758212">K3758212 </a><br> psaC 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758213">K3758213 </a><br> psaJ 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758214">K3758214 </a><br> psbC 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758215">K3758215 </a><br> psbK 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758216">K3758216 </a><br> rbcL 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758217">K3758217 </a><br> rpl20 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758218">K3758218 </a><br> rpl32 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758219">K3758219 </a><br> rps4 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758220">K3758220 </a><br> rps7 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758221">K3758221 </a><br> rps16 3'UTR Os </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758222">K3758222 </a><br> TMV 3'UTR </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758223">K3758223 </a><br> TYMV 3'UTR </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758224">K3758224 </a><br> BMV 3'UTR </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758225">K3758225 </a><br> rrnB 3'UTR Ec </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758226">K3758226 </a><br> atpA 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758227">K3758227 </a><br> atpF 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758228">K3758228 </a><br> ndhJ 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758229">K3758229 </a><br> psbC 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758230">K3758230 </a><br> psbI 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758231">K3758231 </a><br> psbJ 3'UTR Ta </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758232">K3758232 </a><br> rbcL 3'UTR Qr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758233">K3758233 </a><br> rbcL 3'UTR (long) Cr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758234">K3758234 </a><br> rbcL 3'UTR (short) Cr </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758235">K3758235 </a><br> psaC 3'UTR Nt </li>
 +
 
 +
</b>
 +
</ul>
 +
</html>]]
 +
 
 +
 
 +
 
 +
[[File:T--Marburg--3'Homology_Symbol.png|120px|thumb|none|<html>
 +
<ul> <b>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758251">K3758251 </a><br> trnfM 3'Homology Nt </li>
 +
<li> <a href="https://parts.igem.org/Part:BBa_K3758253">K3758253 </a><br> trnA 3'Homology Nt </li>
 +
</ul>
 +
</b>
 +
</html>]]
 +
 
 +
</div>
 +
 
 +
==References==
 +
[1] Aboul-Maaty, N. A.-F., & Oraby, H. A.-S. (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43(1). https://doi.org/10.1186/s42269-019-0066-1
 +
 
 +
[2] Fuentes, P., Zhou, F., Erban, A., Karcher, D., Kopka, J., & Bock, R. (2016). A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. ELife, 5. https://doi.org/10.7554/elife.13664
 +
 
 +
[3] Filée, J., & Forterre, P. (2005). Viral proteins functioning in organelles: a cryptic origin? Trends in Microbiology, 13(11), 510–513. https://doi.org/10.1016/j.tim.2005.08.012
 +
 
 +
[4] Liere, K., & Börner, T. (2007). Transcription and transcriptional regulation in plastids. In Cell and Molecular Biology of Plastids (pp. 121–174). Springer Berlin Heidelberg. https://doi.org/10.1007/4735_2007_0232
 +
 
 +
[5] Xie, G., & Allison, L. (2002). Sequences upstream of the YRTA core region are essential for transcription of the tobacco atpB NEP promoter in chloroplasts in vivo. Current Genetics, 41(3), 176–182. https://doi.org/10.1007/s00294-002-0293-z
 +
 
 +
[6] Kuroda, H., & Maliga, P. (2002). Overexpression of the clpP 5′-Untranslated Region in a Chimeric Context Causes a Mutant Phenotype, Suggesting Competition for a clpP-Specific RNA Maturation Factor in Tobacco Chloroplasts. Plant Physiology, 129(4), 1600–1606. https://doi.org/10.1104/pp.004986
 +
 
 +
[7] Klinkert, B. (2006). Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon. Nucleic Acids Research, 34(1), 386–394. https://doi.org/10.1093/nar/gkj433
 +
 
 +
[8] Zhelyazkova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., & Barkan, A. (2011). Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. Nucleic Acids Research, 40(7), 3092–3105. https://doi.org/10.1093/nar/gkr1137
 +
 
 +
[9] Zoschke, R., Kroeger, T., Belcher, S., Schöttler, M. A., Barkan, A., & Schmitz-Linneweber, C. (2012). The pentatricopeptide repeat-SMR protein ATP4 promotes translation of the chloroplastatpB/EmRNA. The Plant Journal, 72(4), 547–558. https://doi.org/10.1111/j.1365-313x.2012.05081.x
 +
 
 +
[10] Zhang, L., Zhou, W., Che, L., Rochaix, J.-D., Lu, C., Li, W., & Peng, L. (2019). PPR Protein BFA2 Is Essential for the Accumulation of the atpH/F Transcript in Chloroplasts. Frontiers in Plant Science, 10. https://doi.org/10.3389/fpls.2019.00446
 +
 
 +
[11] Tangphatsornruang, S., Birch-Machin, I., Newell, C. A., & Gray, J. C. (2010). The effect of different 3′ untranslated regions on the accumulation and stability of transcripts of a gfp transgene in chloroplasts of transplastomic tobacco. Plant Molecular Biology, 76(3–5), 385–396. https://doi.org/10.1007/s11103-010-9689-1
 +
 
 +
[12] Eberhard, S., Drapier, D., & Wollman, F.-A. (2002). Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. The Plant Journal, 31(2), 149–160. https://doi.org/10.1046/j.1365-313x.2002.01340.x
 +
 
 +
[13] Pfalz, J., Bayraktar, O. A., Prikryl, J., & Barkan, A. (2009). Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts. The EMBO Journal, 28(14), 2042–2052. https://doi.org/10.1038/emboj.2009.121
 +
 
 +
[14] Schaumberg, K. A., Antunes, M. S., Kassaw, T. K., Xu, W., Zalewski, C. S., Medford, J. I., & Prasad, A. (2015). Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nature Methods, 13(1), 94–100. https://doi.org/10.1038/nmeth.3659
 +
 
 +
[15] Stukenberg, D., Hensel, T., Hoff, J., Daniel, B., Inckemann, R., Tedeschi, J. N., Nousch, F., & Fritz, G. (2021). The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens. ACS Synthetic Biology, 10(8), 1904–1919. https://doi.org/10.1021/acssynbio.1c00126
 +
 
 +
[16] Suzuki, J. Y., Sriraman, P., Svab, Z., & Maliga, P. (2003). Unique Architecture of the Plastid Ribosomal RNA Operon Promoter Recognized by the Multisubunit RNA Polymerase in Tobacco and Other Higher Plants. The Plant Cell, 15(1), 195–205. https://doi.org/10.1105/tpc.007914
  
−
[4] Occhialini, A., Piatek, A. A., Pfotenhauer, A. C., Frazier, T. P., Stewart, C. N., Jr., & Lenaghan, S. C. (2019). MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology. Plant Physiology, 179(3), 943–957. https://doi.org/10.1104/pp.18.01220
+
[17] Occhialini, A., Piatek, A. A., Pfotenhauer, A. C., Frazier, T. P., Stewart, C. N., Jr., & Lenaghan, S. C. (2019). MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology. Plant Physiology, 179(3), 943–957. https://doi.org/10.1104/pp.18.01220

Latest revision as of 11:46, 10 August 2023

Prrn16, rrn16 Promoter (-64 to +17) (N. tabacum)


Part Description

This part was created using the Phytobrick Entry Vector with GFP dropout BBa_K2560002 and was designed to be compatible with the Phytobrick assembly standard. This years BioBricks were either constructed via PCR from purified DNA samples using a CTAB based method [1], by primer annealing, primer annealing, and extension reactions or synthesized via IDT/Twist.


All parts this year were produced to be used in the chloroplast of different plant species. For the characterization of these parts they were tested in chloroplast cell-free systems (ccfs) from either the same species or they were tested in ccfs from other plant species. Plastid parts offer the benefit of highly conserved regulatory sequences that can be used across species. Although characterizing chloroplast parts is a huge effort, in literature, it has been shown that plastid parts can be used across species to drive gene expression [2]. We believe that based on this knowledge we can create valuable parts that can be screened for activity in our system with the final goal of building a variety of different parts. This collection shall help combat unwanted recombination events in vivo that sometimes impede the successful functionality of the genetic design.

The PEP Promoter

Transcription in the chloroplast is mainly driven by two different RNA Polymerases: plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP).
The PEP is a bacterial-like polymerase that is a remnant of the chloroplast’s cyanobacterial ancestor and is only capable of promoting gene expression in the plastid. These polymerases are able to interact with nuclear-encoded sigma factors and therefore are able to recognize bacterial promoter motives such as the -35 (TTGACA) and the Pribnow (TATAAT) box. Similar to bacteria there are different sigma factors promoting gene expression under different growth conditions. As the PEP is structurally more sophisticated there are even more peptides involved in DNA transcription that is not fully understood yet.


The NEP Promoter

The NEP is a T3/T7 phage-like polymerase that is encoded in the nucleus and is imported into the chloroplast. It was proposed that this polymerase is a remnant of a horizontal gene transfer from a bacterium to a eubacterial ancestor of today's plant cells [3] [4]. This type of polymerase mainly promotes gene expression in the early developmental stages of the chloroplast. In mature chloroplasts, it continues to transcribe housekeeping genes like the subunits of the plastid-encoded polymerase (rpoA, rpoB, rpoC1, and rpoC2) and proteins involved in fatty acid biosynthesis such as acetyl-CoA carboxylase (accD). In contrast, the PEP is rather active in mature chloroplasts and is primarily involved in the expression of photosynthetic genes. For other non-photosynthetic genes, motives of both polymerases can be found and it has been shown that both can promote transcription using deletion studies of important promoter sequences [5] [6].

Characterization & Measurement

Batch effect

A major difficulty when working with cell free technology is the reproducibility of reliable data generation. While one batch of cell free extract generation might be perfectly suited for efficient protein production, other batches might not perform that well. Because our chloroplast extracts were prepared from leaves, each preparation could have contained distinct compositions of differentiated leaf cells (for example: mesophyll, palisade parenchyma and bundle sheath) from plants that experienced microclimatic variations. This in turns causes the individual difference in expression strength, making it impossible to quantitatively compare data across measurements.

Dual Luciferase Normalization

To tackle the aforementioned issue, we designed our measurement construct to harbor two individual luciferase cassettes. A standardized cassette that includes the Firefly luciferase (FLuc) is included in every measurement construct in order to have a reference point to which we can normalize our gained data to[14] . The second cassette consists of a Nano Luciferase (NLuc) harboring a placeholder part in either the promoter, 5’UTR or 3’UTR position. This enables quick exchange of a series of lvl0 parts allowing for high throughput characterization of genetic parts.

sfGFP_Cell-Free_Tobacco

Figure 1: SBOL Visualization of our Dual Luciferase lvl2 measurement vector
On the left you can see the inverted Firefly luciferase that is used for ratiometric normalization of our data.
The right cassette shows the NanoLuc luciferase, in which Dropout sequences have been included for rapid exchange of parts in either the promoter, 5'UTR or 3'UTR position


Placeholder

For the characterization of the parts produced this year, we made use of Golden Gate placeholder parts introduced by [http://2019.igem.org/Team:Marburg iGEM Marburg 2019]: BBa_K3228060, BBa_K3228061 and BBa_K3228063. These placeholder parts can be used in assemblies to subsequently replace it with another part of the same type in a secondary Golden Gate assembly. A placeholder can rationalize large-scale assemblies: Instead of building each plasmid from scratch, a placeholder is used to generate an entry vector.

Results

Marburg collection 3.0

We proudly present the third expansion of the Marburg collection [15]. The Marburg collection is a Golden Gate based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. The collection overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids. 36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units.

The original [http://2018.igem.org/Team:Marburg/Part_Collection Marburg Collection] contains 123 parts in total, including: inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. The toolbox was constructed as a foundation for future iGEM teams to empower accelerated progression in their ambitious projects.

Our collection includes genetic parts suitable for use in the chloroplast. Among parts from the chloroplast of Nicotiana tabacum, we built parts from the chloroplast of Spinacia oleracea, Oryza sativa,Triticum aestivum and Quercus robur. With our contribution, we aim to accelerate research in the field of plastid engineering.


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]



Overview Marburg collection 3.0



References

[1] Aboul-Maaty, N. A.-F., & Oraby, H. A.-S. (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43(1). https://doi.org/10.1186/s42269-019-0066-1

[2] Fuentes, P., Zhou, F., Erban, A., Karcher, D., Kopka, J., & Bock, R. (2016). A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. ELife, 5. https://doi.org/10.7554/elife.13664

[3] Filée, J., & Forterre, P. (2005). Viral proteins functioning in organelles: a cryptic origin? Trends in Microbiology, 13(11), 510–513. https://doi.org/10.1016/j.tim.2005.08.012

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