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Our Part Collection﹀

Engineering non-model organisms requires discovery of organism-specific parts or re-tuning of parts characterized in model organisms. Our part collection provides modular cloning (MoClo) (Webber et al., 2018) compatible parts that can be used for predictable expression of genes across different bacterial species.

Our collection provides essential parts to overcome the need to identify parts for different bacterial hosts and insulate the parts from variations associated with microbial context.

Identification of origins of replication for different bacterial species is laborious. Although broad host range plasmids can be used across a wide range of bacterial species, they work in specific groups of microorganisms (Jain and Srivastava., 2013). Our toolkit provides all the components (four proteins and two origins of replication) of the Phi29 replication machinery necessary to establish host independent (orthogonal) replication. The advantage of establishing orthogonal replication, is little or no knowledge of bacterial host is needed and higher control of the system can be established.

Non-model bacterial species have a limited collection of promoters (Yang et al., 2018) . Our toolkit provides T7 promoter variants of different strengths which in combination with a T7 RNA polymerase mixed forward loop (UBER system), can be used for gene expression across different bacterial species. In addition, the collection consists of a broad host range promoter (P_BHR) based on conserved regions of -10 region and -35 region sequences in E.coli and B.subtilis (Yang et al., 2018) .

To achieve modularity and predictability of synthetic parts, insulation from their genetic context needs to be established. Sequences downstream of the transcription start site (TSS) of promoters end up in transcripts and are shown to significantly affect translational rates (Lou et al., 2012) . Our toolkit consists of ribozymes which when included in 5’UTR sequences, due to their self cleavage properties, cleave any artifacts left behind in the transcripts upstream of the RBS.

Our toolkit contains an RBS (universal RBS) predicted to work using RBS calculator 2.0 across a range of bacterial species. We have demonstrated the working of the universal RBS in E.coli and P.putida.

Codon usage across bacterial species varies. Our toolkit provides a cross-species codon harmonization tool. The tool can be used to generate coding sequences with equal codon usage across a range of organisms.

Our toolkit consists of variants of T7 terminators. Use of these variants reduces chances of homologous recombination between transcriptional units (Temme et al., 2012) .

Behavior of parts is influenced by microbial context dependent variations (eg. copy number, transcription rates and translation rates) and to overcome the need to re-characterize parts for different bacterial species, an incoherent feed forward loop (iFFL) can be implemented in a genetic circuit (Segall-Shapiro et al., 2018) . To establish an iFFL genetic circuit, TALE (Transcriptional Activator Like Effector) proteins can be used as repressors. Our toolkit contains the gene to express TALE protein which is designed to target a specific DNA sequence. Furthermore, we engineered a series of promoters that can be targeted by the TALE protein.

Part Collection

♥	Name	Type	Description
♥	BBa_K2918034	Coding	Φ29 DNA polymerase (DNAP/p2)
♥	BBa_K2918001	Coding	Φ29 Terminal Protein (TP/p3)
♥	BBa_K2918002	Coding	Φ29 Single Stranded Binding Protein (SSB/p5)
♥	BBa_K2918003	Coding	Φ29 Double Strand Binding Protein (DSB/p6)
	BBa_K2918033	Other	Φ29 Left origin of replication (Ori-L)
	BBa_K2918061	Other	Φ29 Right origin of replication (Ori-R)
♥	BBa_K2918029	Composite	OriL-GFP-Kan-OriR
	BBa_K2918005	Regulatory	Weak T7 promoter
	BBa_K2918006	Regulatory	medium T7 promoter
	BBa_K2918009	Regulatory	medium T7sp1 promoter
	BBa_K2918010	Regulatory	T7sp1 promoter
♥	BBa_K2918054	Regulatory	P_BHR sp1 promoter
	BBa_K2918013	RBS	BBa_B0032 RBS and SarJ
	BBa_K2918014	RBS	Universal RBS
	BBa_K2918035	RBS	BBa_B0032 RBS and RiboJ
	BBa_K2918036	RBS	SarJ and Universal RBS
	BBa_K2918038	RBS	RiboJ and Universal RBS
	BBa_K2918012	Other	Ribo J
	BBa_K2918039	Other	SarJ
♥	BBa_K2918050	Composite	Cross-species based iFFL
♥	BBa_K2918040	Composite	T7 promoter based optimized iFFL
	BBa_K2918046	Composite	T7 promoter based iFFL
	BBa_K2918029	Composite	OriL-GFP-Kan-OriR

Our Best Basic Part﹀
Our best basic part is P_BHRsp1 promoter, BBa_K2918054. We successfully demonstrated its function in Escherichia coli and Pseudomonas putida. Furthermore, it contains a binding site for TALE (Transcriptional Activator like Effector) repressor protein, facilitating it’s implementation in incoherent feed forward loop genetic circuits.
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<a id="bestcomposite" class="toggle" href="javascript:void(0);" >Our Best Composite Part﹀</a>

Our favourite compostie part is <a target="_blank" class="http://parts.igem.org/Part:BBa_K2918050">BBa_K2918050, Cross-species based iFFL</a>. Although many variables play a role in the level of gene expression, we managed to reduce the difference in expression levels by 2 orders of magnitude through the implementation of this iFFL. We therefore set the basis for transferring genetic circuits across bacterial species.

<a id="allparts" class="toggle" href="javascript:void(0);" >All parts﹀</a>

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Name	Type	Description
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918000">BBa_K2918000</a>	Regulatory	P_BHR
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918001">BBa_K2918001</a>	Coding	Φ29 Terminal Protein (TP/p3)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918002">BBa_K2918002</a>	Coding	Φ29 Single Stranded Binding Protein (SSB/p5)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918003">BBa_K2918003</a>	Coding	Φ29 Double Strand Binding Protein (DSB/p6)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918005">BBa_K2918005</a>	Regulatory	Weak T7 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918006">BBa_K2918006</a>	Regulatory	medium T7 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918007">BBa_K2918007</a>	Regulatory	Wild type T7 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918008">BBa_K2918008</a>	Coding	Transcription Activator like Effector protein (TALEsp1)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918009">BBa_K2918009</a>	Regulatory	medium T7sp1 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918010">BBa_K2918010</a>	Regulatory	T7sp1 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918011">BBa_K2918011</a>	Regulatory	P_BHR sp1 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918012">BBa_K2918012</a>	Other	Ribo J
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918013">BBa_K2918013</a>	RBS	BBa_B0032 RBS and SarJ
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918014">BBa_K2918014</a>	RBS	Universal RBS
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918015">BBa_K2918015</a>	Terminator	Wild type T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918016">BBa_K2918016</a>	Terminator	T7 terminator variant
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918017">BBa_K2918017</a>	Composite	0.1 T7 promoter - Universal RBS - Φ29 9 TP - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918018">BBa_K2918018</a>	Composite	0.5 T7 promoter - Universal RBS - Φ29 TP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918019">BBa_K2918019</a>	Composite	WT T7 promoter - Universal RBS - Φ29 TP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918020">BBa_K2918020</a>	Composite	0.1 T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918021">BBa_K2918021</a>	Composite	0.5 T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918022">BBa_K2918022</a>	Composite	WT T7 promoter - Universal RBS - Φ29 DNAP - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918023">BBa_K2918023</a>	Composite	Weak T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918024">BBa_K2918024</a>	Composite	Medium T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918025">BBa_K2918025</a>	Composite	WT T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918026">BBa_K2918026</a>	Composite	0.1 T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918027">BBa_K2918027</a>	Composite	0.5 T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918028">BBa_K2918028</a>	Composite	WT T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918029">BBa_K2918029</a>	Composite	OriL-GFP-Kan-OriR
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918030">BBa_K2918030</a>	Composite	WT T7 promoter - Universal RBS - GFP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918031">BBa_K2918031</a>	Composite	0.1 T7 promoter - Universal RBS - GFP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918032">BBa_K2918032</a>	Composite	0.5 T7 promoter - Universal RBS - GFP - T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918033">BBa_K2918033</a>	Other	Φ29 Left origin of replication (Ori-L)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918034">BBa_K2918034</a>	Coding	Φ29 DNA polymerase (DNAP/p2)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918035">BBa_K2918035</a>	RBS	BBa_B0032 RBS and RiboJ
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918036">BBa_K2918036</a>	RBS	SarJ and Universal RBS
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918037">BBa_K2918037</a>	Coding	Cross-species harmonized eGFP
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918038">BBa_K2918038</a>	RBS	RiboJ and Universal RBS
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918039">BBa_K2918039</a>	Other	SarJ
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918040">BBa_K2918040</a>	Composite	T7 promoter based optimized iFFL
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918041">BBa_K2918041</a>	Coding	Kanamycin resistance gene
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918043">BBa_K2918043</a>	Composite	T7sp1-RiboJ-UniRBS-hGFP-T7 terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918044">BBa_K2918044</a>	Composite	T7sp1-RiboJUniRBS-jGFP-T7terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918045">BBa_K2918045</a>	Composite	Pbhrsp1-RiboJUniRBS-jGFP-T7terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918046">BBa_K2918046</a>	Composite	T7 promoter based iFFL
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918047">BBa_K2918047</a>	Regulatory	low T7sp1 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918048">BBa_K2918048</a>	Composite	0.5 T7 promoter based iFFL
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918049">BBa_K2918049</a>	Composite	Pbhrsp1_v2, RiboJ_URBS_jGFP_T7terminator
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918050">BBa_K2918050</a>	Composite	Cross-species based iFFL
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918051">BBa_K2918051</a>	Composite	Pbhr_sp2, RiboJURBS, jGFP,T7
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918052">BBa_K2918052</a>	Coding	eGFP
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918053">BBa_K2918053</a>	Regulatory	T7 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918054">BBa_K2918054</a>	Regulatory	P_BHR sp1 promoter
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918055">BBa_K2918055</a>	Regulatory	P_BHR promoter sp2
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918061">BBa_K2918061</a>	Other	Φ29 Right origin of replication (Ori-R)
<a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2918062">BBa_K2918062</a>	Composite	Universal RBS (adapted for modular cloning with ribozymes)

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References

<a id="Jain" href="https://academic.oup.com/femsle/article/348/2/87/731695" target="_blank"> Jain, A. and P. Srivastava (2013). "Broad host range plasmids." FEMS Microbiology Letters 348(2): 87-96. </a>
<a id="Lou" href="http://www.ncbi.nlm.nih.gov/pubmed/23034349" target="_blank"> Lou, C., Stanton, B., Chen, Y., Munsky, B., & Voigt, C. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137-1142. doi: 10.1038/nbt.2401 </a>

<a id="Segall-Shapiro" href="http://doi.org/10.1038/nbt.4111" target="_blank"> Segall-Shapiro, T. H., et al. (2018). "Engineered promoters enable constant gene expression at any plasmid copy number in bacteria." Nature Biotechnology 36: 352. </a>
<a id="Temme" href="https//academic.oup.com/nar/article/40/17/8773/2411560" target="_blank"> Temme, K., Hill, R., Segall-Shapiro, T., Moser, F., & Voigt, C. (2012). Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Research, 40(17), 8773-8781. doi: 10.1093/nar/gks597 </a>

<a id="Yang" href="http://www.ncbi.nlm.nih.gov/pubmed/29061047" target="_blank"> Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." ACS Synthetic Biology 7(1): 287-291. </a>
<a id="Webber" href="http://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. </a>

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                  <ul class=" nav nav-pills nav-stacked accordion" data-spy= "affix" data-offset-top="180">
                      <li><a class="jump" href="#partcollection" onclick='$("#partcollection").click()'>Our Part Collection</a></li>
-                     <li><a class="jump" href="#bestpart" onclick='$("#bestpart").click()'>Our Best Part</a></li>
+                     <li><a class="jump" href="#bestpart" onclick='$("#bestpart").click()'>Our Favorite Basic Part</a></li>
-                     <li><a class="jump" href="#bestcomposite" onclick='$("#bestcomposite").click()'>Our Best Composite Part</a></li>
+                     <li><a class="jump" href="#bestcomposite" onclick='$("#bestcomposite").click()'>Our Favorite Composite Part</a></li>
 <li><a class="jump" href="#allparts" onclick='$("#allparts").click()'>All Parts</a></li>
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                <p>
-Our best basic part is P<sub>BHR</sub>sp1 promoter, BBa_K2918054. We successfully demonstrated its function in <i>Escherichia coli</i> and <i>Pseudomonas putida</i>. Furthermore, it contains a binding site for TALE (Transcriptional Activator like Effector) repressor protein, facilitating it’s implementation in incoherent feed forward loop genetic circuits.
+Our best basic part is <html><a href="http://parts.igem.org/Part:BBa_K2918054">P<sub>BHR</sub>sp1 promoter, BBa_K2918054</a></html>. We successfully demonstrated its function in <i>Escherichia coli</i> and <i>Pseudomonas putida</i>. Furthermore, it contains a binding site for TALE (Transcriptional Activator like Effector) repressor protein, facilitating it’s implementation in incoherent feed forward loop genetic circuits.
                          <br>