Synthetic Peptides - BBa_K1152000 - 2005

We provide the constructs for the novel Dipeptide- and Tripeptide-Synthetases. Furthermore, we submitted the single modules that they are comprised of. These parts should serve as the basis of a future library for standardized work with NRPS. The modules are specific for different amino acids: Phenylalanine (tycA), Proline (tycB1) and Leucine (tycC6).

Indigoidine-Tag - BBa_K1152006 & 2007

One of our major achievements is the establishment of an easily detectable, inert and universal tag - the Indigoidine-Tag. With this tag, purification and validation of novel NRPs is significantly eased. The characteristics of the tag compare to those of the GFP-tag for proteins. We submit the helper-construct for tagging any desired Non-Ribosomal Peptide as our favorite part.

Tag-Optimization - BBa_K1152008 & 2013-2019

Besides establishing the Indigoidine-Tag, we focussed on optimizing its functionality and efficiency. Therefore, we modified the natural sequence of the Indigoidine synthetase indC by domain exchange which lead to altered and in some cases enhanced efficiency of the NRP-production which could be measured by optical density. We submit the collection of alternate T-domains, of which we created several functional, synthetic ones as Best Part Range.

Tag-Characterization - BBa_K1152009 & 2012

During Tag-Optimization, we also focussed on the enzymes that are required for the activation ...

Natural Parts

We offer several natural parts to the library. Firstly, three modules from the Tyrocidine cluster from B. parabrevis that are specific for three different amino acids: BBa_K1152000 (tycA) for Phenylalanine, BBa_K1152001 (tycB1) for Proline and BBa_K1152003 (tycC6) for Leucine. Secondly, we have submitted the Indigiodine synthetase indC from P. luminescens (cds: BBa_K1152008, integratable device: BBa_K1152013). And thirdly, we offer a collection of PPTases (see image) which are required enzymes in order to activate NRPSs and turn them from the apo- into the hoho-form: BBa_K1152009, BBa_K1152010, BBa_K1152011, BBa_K1152012.

We propose BBa_K1152013 as Best new BioBrick Part, Natural, because it encodes the original NRPS module from Photorhabdus luminescens capable of synthesizing Indigoidine, which offers the opportunity to combining it with modules from other NRPSs to yield fusion peptides with a blue pigment tag. Accompanying this BioBrick, we propose two standards (RFC 99 & RFC 100) and established protocols which allow for BioBrick assembly or Gibson assembly and through this to a high-throughput creation of synthesized, tagged peptides. Besides this, Indigoidine is belived to have antimicrobial and anti-cancerous effects.

We used Indigoidine in the subprojects that were dealing with the tagging of NPRs and, in the course of this, characterized its functionality thoroughly. Hence more information on Indigoidine is available on the Peptide-Tagging page and the Tag-Optimization page.

Engineered Parts

We built different BioBricks in the course of investigating the modularity of NRPS. We offer two novel Non-Ribosomal Peptide Synthetases, one which is an assembly line for a Proline-Leucine Dipeptide (BBa_K1152004), the other one is a synthetase for a Phenylalanine-Ornithine-Leucine Tripeptide (BBa_K1152005). You can learn more about these parts on our Synthetic Peptides page.

Besides this, we created an inter-species fusion-peptide of the TycC4-module and the Indigoidine synthetase IndC, thereby giving a proof of concept for both, inter-species flexibility of modules and domains, and tagging of short peptides with the blue pigment Indigoidine. This is explained in detail on our Peptide-Tagging page and in the parts' registry: BBa_K1152006.

Furthermore, we created several synthetic variants of the IndC-module by introducing a T-domain from another synthetase or by introducing entirely synthetic T-domains: BBa_K1152015, BBa_K1152016, BBa_K1152017, BBa_K1152018, BBa_K1152019, BBa_K1152020. We propose this set of parts as Best Parts Collection as they represent a valuable ensemble of various T-domains with different properties and besides this, proof the extent of flexibility of NRPS. Being designed semi-rationally based on multiple sequence alignments, we discovered that modified IndC-variants exhibit distinct synthesis rates for Indigoidine depending on the T-domains of this part collection. We therefore used them for Optimization of the Indigoidine-Tag.

During our work with NRPS, we did not only create several synthetic parts in order to characterize and improve the efficiency of Indigoidine and synthesize new peptides, but also improved the parts BBa_K802006 and BBa_K302010, which encode for sfp (a PPTase) from Bacillus subtilis but were deviating in three amino acids from the sequence published on National Center for Biotechnology Information (NCBI). We therefore propose BBa_K1152009 as Most Improved Registry Part, because it encodes for sfp, a 4'-Phosphopanthetheinyl-transferase (PPTase) crucial for the activation of T-domains and contains the aforementionned three divergent amino acids. It is the first BioBrick in the registry that is explicitly encoding sfp, and it is annotated and characterized. Learn more about PPTases and their characterization on the Tag-Optimization page!

Engineered Devices

The work mentionned above was significantly eased by two helper constructs that we designed and we would hence like to promote them to the entire community. As it is the central idea of our project to make the very potent NRPS-system accessible to the iGEM community, we believe that those two parts are of a very high value.

We are therefore proud to promote our favorite part, namely BBa_K1152007, which is a helper construct for NRP-Indigoidine-tagging. In order to tag peptides with Indigoidine, a C-domain has to be inserted in front of the IndC-module, in order to standardize this, and in order to allow anyone to create tagged constructs easily and with low background, we developed this ccdB-driven (as negative selection), highly efficient (i.e. 0 % false-positive rate) cloning tool. The plasmid is compatible with custom non-ribosomal peptide (NRP) synthesis due to the standardized C-domain from B. parabrevis. So in summary BBa_K1152007 provides optimized fusion of the NRP to Indigoidine that serves as a tag that eases detection of positive clones that synthesize the desired construct. We therefore propose BBa_K1152007 as Best new BioBrick Part or Device, Engineered, as mentioned above. We found it very helpful to work with this part in the Peptide-Tagging project.

Additionally, we provide the community with the helper-construct that we used for the T-domain shuffling. It is also ccdB-based and therefore minimizes background during Tag-Optimization: BBa_K1152014