THE MOCLO MANIA COLLECTION
Welcome to the heart and soul of our project - our MoClo Mania collection!
We have designed and built this versatile collection from the ground up and are very proud to introduce you to it. All of the genetic parts within our collection can be assembled according to the Modular Cloning system established by Weber et al. in 2011. Furthermore, all of our parts allow for recombinant protein expression in the protozoan parasite Leishmania tarentolae. Why is this a cool thing? Because Leishmania have recently been emerging as promising novel expression hosts, especially for the production of recombinant glycoproteins! Due to their intricate protein processing capabilites that closely resemble those of mammalian cells, no other microbial expression host recreates complex, human-like post-translational modifications better than Leishmania!
We believe in Leishmania’s potential as a powerful and valuable expression host! That’s why we want to facilitate protein production in Leishmania and make it more approachable and feasible for researchers and iGEM-teams everywhere! By establishing the Modular Cloning system in Leishmania, we can promote the easy and efficient generation of variable genetic constructs ready for transfection and transgenic expression. It means that all the parts of our collection can be mixed, matched and assembled into constructs with the help of just two enzymes in a simple, one-step, one-pot reaction. We have worked out a variety of protein tags, such as a secretion tag, fluorescent tags, antibody as well as affinity tags that will help researchers and future iGEM-teams to easily modify their protein of interest, express it in Leishmania and make it accessible to downstream detection and purification procedures. Along with our parts collection, our wiki provides a lot of helpful information on how to make a target protein sequence ready for Modular Cloning and how to work with Leishmania cell culture. This way we want to make sure that really everybody can profit from Leishmania's favorable protein processing capabilities!
On the remainder of this page, we want to take the time and introduce you to our Modular Cloning approach as well as to the different basic parts that belong to our MoCloMania collection. You can find a quick overview over all of them in the table below. Keep in mind that many of the parts were designed for different cloning positions, meaning that they can, for example, work as an N-terminal as well as a C-terminal protein tag. In the paragraphs below, you can find more detailed information on the functionality and history of the parts. In order to find out more about their experimental use, just click on the highlighted cloning positions and it will take you to that part’s registry page.
MODULAR CLONING APPROACH
|N-terminal tag||coding sequence 1||coding sequence 2||C-terminal tag|
|SARS-CoV-2 RBD||SARS-CoV-2 RBD||3xHA|
As mentioned before, we have chosen the Modular Cloning system for our expression approach because it allows for easy and efficient generation of variable genetic constructs in simple, one-pot reactions. The Modular Cloning (MoClo) system as established by Weber et al. relies on the two executive type IIS restriction enzymes BsaI and BbsI for assembly of basic genetic units called L0 parts into composite genetic constructs called L1. These L1 parts form a cohesive transcriptional unit consisting of promoter,coding sequence and terminator. In order to fill these different genetic attributes with the sequences of choice (e.g. a preferred promoter or a target gene), the L1 reading frame is divided into discrete cloning positions, e.g. A2 or B5, see Figure 1.
Each of these cloning positions is flanked by a specified set of four nucleotide overhangs that are generated upon BsaI digestion of the basic parts and that mediate the coordinated assembly of all basic parts into a L1 composite construct in the pre-termined order. This fixed order given by the specified overhang system is what allows for MoClo restriction and ligation to happen simultaneously in one single reaction. It also promotes the great modularity of the MoClo cloning system, since every basic part is specifically adapted to a single cloning position and can be exchanged for other parts occupying the same cloning position at will. In our MocloMania collection, we only rely on cloning positions B2 - B5 for our L1 assembly. As seen in Figure 1, these cloning positions code for the actual target protein sequence, including N-terminal and C-terminal tags. Our system does not include any promoters or terminators, because these genetic units are already stably included in our L1 expression vector weird_plex.
Beyond L1 constructs, Modular Cloning also allows for assembly of several complete transcriptional units into a multi-gene construct called L2. This can be handy for fusing a target protein transcription unit with a selection marker or a resistance cassette for selective growth of the transfected expression host cells. Since our L1 expression vector already contains an E. Coli expression cassette for propagation in E. Coli as well as a bleomycine resistance for selection of sucessfully transfected Leishmania, our cloning systedoesn't require further extension into L2 assembly. We hope that this gave you a good introduction into the MoClo system and as to what cloning positions we utilize and why. As always, if you have any further questions, please feel free to reach out to us via our e-mail or social media!
In silico elimination of SapI restriction sites
It was our supervising research group that introduced us to the MoClo system after Weber et al. We soon discovered its advantages over conventional restriction cloning and therefore decided to make this cloning system the genetic base to our project. As mentioned before, the Weber-based Modular Cloning system is based on the type IIS restriction enzymes BsaI and BbsI. Thus, the basic parts of our MocloMania collection> were domesticated against BsaI and BbsI. Our basic parts are specifically adapted towards our Modular Cloning approach and are codon optimized towards Leishmania tarentolae. We thus highly recommend using them within the framework of our MocloMania expression system. Therefore we didn't see sense in domesticating them towards type IIS loop assembly, a cloning standard not related to our project. Thus, some of our basic part sequences contain SapI restriction sites.
However, this doesn't comply with the iGEM RCF 1000 cloning standard, the only cloning standard available in the iGEM Registry for type IIS based cloning systems. We are proud of the work we have done over the past year and feel that our implemented parts are valid despite incompatibility to type IIS loop assembly. But we were afraid that pages of incompatible parts might be entirely avoided by the judges and wanted to make sure that our parts would not be outruled without being reviewed. Thus, we consciously decided to eliminate any SapI restriction sites occurring in our basic parts. The parts affected by this alteration are all our basic parts coding for the SARS-CoV-2 receptor binding domain, the GST fusion tag as well as our L1 expression vector weird_plex. The restriction sites were eliminated by introducing silent point mutations that were noted in the respective part's page so that interested researchers can choose either to use the uploaded sequence for Loop Assembly or the original sequence for Modular Cloning.
Since Modular Cloning after Weber is a popular cloning system in many labs nowadays and we have heard from other iGEM-teams with similar issues, we would suggest that both MoClo based approaches as well as Loop assembly are acknowledged as two separate cloning standards accepted in the iGEM Registry. We realize that it is difficult to incorporate all different type IIS cloning systems out there into a comprehensive standard, but we think that incorporating a wider variety of type IIS approaches into the Registry requirements will facilitate part registry for many future iGEM-teams to come and help to diversify and advance the Part Registry even more towards iGEM's goal - open scientific communities sharing modular parts that make synthetic biology more accessible and feasible for everyone.
The protein with the poetic name mVenus is a yellow fluorescent protein and a derivation of the famous green fluorescent protein GFP, originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962.  Even though the purpose of this extraordinary sea creature‘s bioluminescence is still unknown, its fluorescent proteins are of immense importance to modern biological research. This is not surprising, considering the great practicability of fluorescent reporter systems. By coupling a protein of interest with a fluorescent tag such as mVenus, the target protein can be easily and non-invasively detected within living cells as well as cell preparations with the help of fluorescence spectroscopy and microscopy.
Much like mVenus, the cyan fluorescent protein mCerulean is derived from the green fluorescent protein GFP originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962. Like its molecular siblings, it can be cloned to a gene of interest and thus provide fast and non-invasive screening opportunities for the resulting fusion protein, for example via fluorescence spectroscopy or microscopy.
The 8His-tag is a so-called polyhistidine-tag, meaning that it is a small amino acid chain consisting of, in this case, eight consecutive histidine residues. The histidine sidechain contains a heterocyclic imidazole ring which is negatively charged in neutral to basic conditions and can coordinate with metal ions with very high affinity. Thus, equipping a target protein with a terminal histidine rich peptide sequence allows for purification of the protein via immobilized metal ion affinity chromatography. Here, divalent nickel or cobalt ions are attached to a carrier material and bound by the polyhistidine-tag upon addition of the cell lysate. Meanwhile, non-tagged proteins only display weak metal ion affinity and can be discarded in the flow through. Later on, the bound target protein can be easily eluted from the carrier material with the help of pH titration or high excess molecular imidazole which competes with the His tag for metal ion coordination. Since His tags are small in size and rely solely on the protein’s primary structure, they are the preferred choice for protein purification under denaturing conditions. The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to a Strep-tag II.
When it comes to biomolecular detection and purification set-ups, the Strep-tag system is an absolute laboratory staple. In its principle it relies on the high affinity binding of the homo-tetrameric protein streptavidin, first isolated from the bacterium Streptomyces avidinii, to the vitamin biotin. The binding of these two biomolecules is one of the strongest non-covalent interactions observed in nature and inspired the creation of many derivatives with even further enhanced binding capabilities. The German biotech company IBA Life Sciences developed a synthetic eight amino acid peptide called Strep-tag II which binds to the specifically engineered streptavidin derivative Strep-Tactin with high specificity. Thus, fusing a gene of interest to a Strep-tag II allows for efficient purification of the target protein by affinity chromatography over immobilized Strep-Tactin. The bound target protein can later be eluted with desthiobiotin. Due to its small size and biochemical inertia, the addition of a Strep-tag usually doesn’t influence correct target protein folding and functionality. Furthermore, the purification procedure can be executed under physiological conditions, making the Strep-tag system particularly suitable for purification of native, functional proteins. The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to an 8His-tag.
The GST-tag has been around since the late 1980s and, unlike most other purification tags, does not only consist of a small peptide, but rather an entire functional protein. GST is short for glutathione S-transferase, a highly abundant cytosolic enzyme that is usually involved in protecting prokaryotic as well as eukaryotic cells from extracellular toxins. It does this with the help of the redox peptide glutathione, to whose reduced form it binds with very high affinity. By fusing a gene of interest with a GST-tag, the resulting fusion protein can be purified from cell preparations with the help of affinity chromatography, employing reduced glutathione (GSH) as the respective column material. Due to the highly specific binding, only GST-tagged target protein should remain bound to the column and can afterwards be eluted by adding high excess molecular GSH. Since the affinity binding relies on the structural integrity of the GST protein, GST-tag purification can only be performed under native, non-denaturing conditions.  The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to a TEV protease recognition site.
The HA epitope tag was first established in 1988 by J. Field and his colleagues, making it one of the oldest protein tags still in use. Its characteristic amino acid sequence YPYDVPDYA is based on a small segment of the viral protein hemagglutinin (abbreviated as HA), one of three integral membrane proteins in the Influenza A virus. The epitope segment was chosen due to its high immunogenicity and antibody affinity which is further increased by fusing multiple epitope copies in a row (three copies = 3xHA). A vast variety of monoclonal primary antibodies from different species have been established against the HA epitope, eliminating the need for expensive protein-specific antibodies and facilitating target protein detection and purification.
size | 2.5 kDa
available in cloning position B3
The Myc-tag, along with 3xHA and 3xFLAG-tag, is yet another epitope tag designed for protein purification and detection with the help of specific antibodies. It consists of a small peptide whose amino acid sequence (EQKLISEEDL) was originally derived from the transcription factor MYC, which is encoded by the human protooncogene c-myc and plays an important role in the regulation of endogenous gene expression. A pathological, constitutive expression of c-myc may lead to an increased gene expression of genes involved in cell cycle regulation and cell proliferation, thus facilitating the formation of cancer. The gene’s and with it the tag’s name stems from the cancerous disease myelocytomatosis which is observed in birds and is caused by a virus-induced misregulation of c-myc gene expression. When it comes to experimental uses, the Myc-tag can be used for all detection and purification purposes that involve binding of the target protein to antibodies, such as immunoprecipitation or affinity chromatography, and eliminates the need for protein-specific antibodies. This basic part is fused to an HRV 3C protease recognition site.
size | 4.2 kDa
available in cloning position B3
Much like the 3xHA tag, the 3xFLAG®-tag is a small polypeptide epitope tag. Yet, unlike the 3xHA-tag, its amino acid sequence is not derived from a naturally occurring protein, but was instead artificially designed to be an epitope for specifically developed primary antibodies. It was first described in 1988 by Hopp et al. as a single Flag tag consisting of the eight amino acid sequence DYKDDDDK. The high content of polyvalently anionic amino acids (like aspartic acid and tyrosine) makes it less likely to interfere with target protein activity. Over the years, many variations of the original Flag-tag were established, including the very commonly used 3xFLAG®-tag patented by SigmaAldrich with the slightly modified sequence DYKDHDG-DYKDHDI-DYKDDDDK. It contains multiple epitope copies, hereby further increasing the tag’s affinity to the anti-Flag antibodies. The 3xFLAG®-tag can be used for all detection and purification purposes that involve binding of the target protein to antibodies, such as immunoprecipitation or affinity chromatography, and eliminates the need for protein-specific antibodies. This basic part is fused to an HRV 3C protease recognition site.
size | 2.3 kDa
available in cloning position B2
The sAP tag is a secretion signal peptide derived from the secretory protein secreted acid phosphatase 1 (sAP1) which was originally purified from Leishmania mexicana, a human pathogenic Leishmania strain. The secretion of acid phosphatases makes the parasites more resistant to oxidative stress induced by the host’s immune cells. The secretion signal peptide found in sAP1 mediates the exocytosis of the protein after its biosynthesis in the endoplasmatic reticulum (ER). This is extremely useful for recombinant protein production, since target proteins can be harvested directly from cell culture supernatant without the need for cell lysis. Since posttranslational glycosylation of proteins happens within ER and Golgi apparatus along the secretory pathway, the sAP1 signal peptide tag furthermore enables the production of glycosylated proteins in Leishmania. This makes the sAP1 tag a vital part of our cloning kit, since employing Leishmania as an alternative expression host for biopharmaceuticals relies on correct reconstruction of the proteins‘ glycosylation patterns.
OUR PROTEIN OF INTEREST
The COVID-19 pandemic has been sweeping the world for almost two years now, costing millions of lives and causing immense economical, cultural and social damage. The virus responsible for the outbreak of the eponymous respiratory disease was named severe acute respiratory syndrome coronavirus 2 or short SARS-CoV-2. Much like other coronaviruses, its infectiousness relies on multimeric protein structures that project outwards from the virus envelope’s outer surface. Due to their appearance, these protein complexes are often called spikes. They consist of a trimeric complex of the so called spike protein and coordinate the recognition, binding and entry of the virus into the host’s cells. The spike protein itself is a glycoprotein consisting of two separate regions, S1 and S2, of which S1 contains the receptor binding domain (RBD) and mediates cell binding, while S2 contains a fusion peptide and initiates the actual endocytosis of the virus particle. As the entry point into host cells, Sars-CoV-2 makes use of the angiotensin-converting enzyme 2 (ACE2), a transmembrane protein mainly present in intestinal, kidney and cardiac cells and involved in blood pressure regulation. The highly specific binding of the Sars-CoV-2 receptor binding domain to ACE2 is significantly dependent on the posttranslational glycosylations that the RBD undergoes. This makes the recombinant production of correctly glycosylated spike protein very valuable for infectiological research and vaccine development. Thus, by including the Sars-CoV-2 RBD into our cloning kit, we not only demonstrate the significance of our expression system to the advancement in current coronavirus research, we also reveal the glycosylation capabilities of our expression host Leishmania tarentolae.
recognition motif | *NLYFQ ▼S, * = E,G,A,M,C,H
fused to B5 GST
The TEV protease is a cysteine protease domain originally derived from the tobacco etch virus (TEV), a plant virus that infects a wide variety of nightshades and weeds, including the eponymous tobacco plant. The protease is known for its extremely high sequence specificity, meaning that it only cleaves proteins upon recognition of a fixed set of amino acid sequences, with little to no off-target effects. This makes it a valuable tool in modern biotechnological research, since it allows for precise protein cleavage at easily predefined positions. By including a TEV recognition motif on either side of a tag sequence, the respective tag can be cleaved off after protein purification via the addition of TEV protease to the mix. Many commercially available TEV proteases are furthermore fused to an affinity tag themselves, making the retrieval of cleaved target protein even more feasible and minimizing the risk of uncoordinated proteolytic activity due to overexposure. The only requirement for a target protein to be fused with our B5 GST-tag and purified using TEV protease is that the protein sequence ought to be free of any intrinsic TEV recognition sequences. This should be ensured beforehand, via sequence analysis or PCR, and any observed recognition sequences should be removed, i.e. by the introduction of silent single point mutations.
HRV 3C Protease
recognition motif | LEVLFQ ▼GP
fused to all B3 parts
HRV 3C is short for human rhinovirus 3C protease (HRV 3C), a cysteine protease that usually helps rhinoviruses to infect human cells by disrupting the cells‘ endogenous transcription activity. By including a HRV 3C recognition motif on either side of a tag sequence, the respective tag can be cleaved off after protein purification via the addition of the protease to the mix. Most commercially available HRV 3C proteases are furthermore fused to an affinity tag themselves in order to facilitate the retrieval of cleaved target protein. The only requirement for a target protein to be fused with an HRV 3C tag and purified using HRV 3C protease is that the protein sequence ought to be free of any intrinsic HRV 3C recognition sequences. This should be ensured beforehand, via sequence analysis or PCR, and any observed recognition sequences should be removed, i.e. by the introduction of silent single point mutations.
L1 expression vector
L1 plasmid backbone
transfection vector for expression in Leishmania tarentolae
At the very bottom of this page, we want to take the time to talk about the real star of the MoClo Mania show - weird_plex. Don't be fooled by its odd-looking name! This plasmid is a real powerhouse. It functions both as a L1 destination vector for Modular Cloning assembly of basic parts and as an expression vector for recombinant protein expression in Leishmania tarentolae. The plasmid that cost us countless hours of labwork to domesticate. The plasmid that makes the implementation of our project, MoClo in Leishmania, possible in the first place. But what is this miraculous plasmid and where does it come from?
Well, first up. We didn't build this plasmid from the ground up. That would have gone way beyond the scope of our project. Its sequence is based on the commercially available Leishmania expression vector pLEXSY_I-blecherry3 distributed by german biotech company Jena Bioscience in their LEXSinduce3 Expression Kit. Jena Bioscience has specialized on selling Leishmania-adapted expression kits that facilitate recombinant gene expression in the protozoan expression host. They offer a variety of plasmids that differ in their genetic make-up and biological properties. While some remain within the cells' cytosole after transfection, resulting in episomal gene expression, others integrate into Leishmania's genome. The pLEXSY_I-blecherry3 plasmid is such an integrative expression vector.
In order to introduce a gene of interest into pLEXSY_I-blecherry3, the vector is equipped with several multiple cloning sites. This insertion region is controlled by a T7 promoter, a DNA sequence specifically bound by the constitutively expressed T7 RNA polymerase. T7 polymerase is an E. Coli phage derived polymerase that, together with its promoter, mediates strong gene expression.  Another cool feature of the pLEXSY_I-blecherry3 plasmid is its inducible gene expression. This is achieved by a downstream tet operator which is part of a tetracycline-controlled activation system commonly found in eukaryotic expression hosts.  Together with the constitutively expressed tet repressor, this operon suppresses binding of the T7 polymerase to its promoter, but can easily be de-activated by the addition of tetracycline to the culture medium. This powerful expression set-up is very cool and make pLEXSY_I-blecherry the perfect plasmid backbone to our MoClo Mania chassis! Why not just use it as is? Well, the multiple cloning sites are great, but they require conventional restriction cloning which can be tedious and time-consuming. Facilitating the gene insertion process by domesticating pLEXSY_I-blecherry3 towards our Modular Cloning system was one of the core objectives of our project.
For post-transfection screening, the pLEXSY_I-blecherry3 plasmid contains a bleomycin resistance that acts as an important selection marker for successful uptake of the L1 expression vector. Another interesting feature - the bleomycine resistance gene is directly fused to an mCherry coding gene. Since both these genes are located downstream of the insertion region and thus under control of the T7 promoter, the production of mCherry correlates with the production of target protein. Monitoring cellular mCherry content can thus be used to determine successful induction and productive target gene expression. This screening process can happen as easily as looking at the plate with your bare eye and picking the colony with the most evident red coloration.
Beyond all of this, the plasmid's cloning frame also includes a Leishmania mexicana derived secretion signal peptide as well as a C-terminal Hexa-histidine-tag, flanked on either end by multiple cloning sites. Thus, pLEXSY_I-blecherry3 allows for either cytosolic or secretory protein expression and optional His-tagging of the target protein. During domestication of the plasmid towards the MoClo system, these two features were eliminated. Instead, their sequences were adapted and turned into functional L0 basic parts within the MoClo Mania collection, the sAP secretion tag and the Strep8His-tag. This way, switching between cytosolic and secretory protein expression is easily coordinated within a simple MoClo reaction.
Additional to all the genetic optimizations towards expression in Leishmania, pLEXSY_I-blecherry3 also contains an E. Coli expression cassette that consists of an ampicillin resistance gene as well as a high copy number E. Coli origin of replication. They allow for the insert-carrying vector to be transformed and amplified in E. Coli which is important because transfection success relies on the input of sufficient DNA amounts. The E. Coli expression cassette is designed to be cleaved off by the enzyme SwaI prior to transfection, leaving behind a linear DNA fragment containing all the genes relevant for transgenic protein expression in Leishmania, including your inserted target sequence.
In order to make the pLEXSY_I-blecherry3 vector suitable for our Modular Cloning approach, it first had to be domesticated towards our cloning system. This was done by eliminating three endogenous BsaI restriction sites via the introduction of single point mutations. This step turned out to be very time-consuming, but it had to be done, since BsaI is one of the two executive type IIS restriction enzymes in the Modular Cloning system and is used for all L1 assemblied. Had the endogenoues restriction sites remained intact, our expression vector would have been cut up into pieces every time we tried to ligate a construct! Furthermore, pLEXSY_I-blecherry3's multiple cloning sites were used to introduce a LacZ-alpha gene cassette into the vector backbone. This way, when the plasmid is transformed into a LacZ-omega carrying E. Coli strain and grown on IPTG/XGAL agar plates, it will result in blue appearing colonies for which can easily be screened with the bare eye. Furthermore, this LacZ-alpha insertion allowed for new BsaI restriction sites to be introduced into the plasmid, flanking either end of the LacZ-alpha gene in inverted orientation. These restriction sites take on the function of the multiple cloning sites in conventional cloning because they enable the insertion of MoClo L1 constructs into our vector.
With the help of all these domestication steps we were able to turn pLEXSY_I-blecherry3 into weird_plex, a jack of two trades. Any basic part within our MoClo Mania collection can be assembled into a cohesive L1 construct and introduced into weird_plex for recombinant expression in one single MoClo reaction. We are very proud and excited to share this novel approach to protein expression in Leishmania with the world and we are confident that it will facilitate the research work of all scientists and iGEM-teams interested in expressing complex glycoproteins and biopharmaceuticals!
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