Plasmid_Backbone

Part:BBa_K3781102

Designed by: Nicolas Bayer   Group: iGEM21_TU_Kaiserslautern   (2021-10-09)


pICH41258

MocloMania

This basic part categorizes as a plasmid backbone and renders the commercially available, pUC19-derived E. Coli expression plasmid named pICH41258. It was originally created by Weber et al. in 2011 and is part of a plasmid library specifically adapted to Modular Cloning assembly. Within the Modular Cloning system, it functions as a Level 0 plasmid backbone for genetic parts destined to occupy the B3 cloning position.[1]

pICH41258 consists of a high-copy number E. Coli origin of replication as well as a gene mediating spectinomycin resistance and a lacZ-alpha gene that enables blue-white screening of successful clones. These features make it suitable for fast and efficient replication in transformed E. Coli.

What sets this plasmid apart from other conventional bacterial expression vectors is that its lacZ-alpha region is flanked by two invertedly oriented BbsI restriction sites. Upon digestion of the plasmid with BbsI, they generate a specified set of four nucleotide overhangs that match the overhangs found in a B2-destined MoClo basic part. Thus, if both part and plasmid are digested in a one-tube reaction, the basic part will insert into the plasmid in its pre-defined orientation. Upon part insertion, the lacZ-alpha fragment is irreversibly replaced, so resulting colonies can be easily screened for the desired genetic construct. Furthermore, the BbsI recognition sites flanking the lacZ-alpha fragment are facing outward, meaning that they themselves are located within the part that will be excised. Thus, once your part has inserted into the plasmid backbone, it cannot be re-digested and get lost again, allowing for restriction and ligation to happen simultaneously.

pICH41258, along with all other plasmid backbones in the Weber library, isn't only designed to host basic gene parts, it also functions as the transient vector for their assembly into larger genetic constructs. On the plasmid, just outside of the lacZ-alpha insertion region, BsaI restriction sites are cleverly placed in direct relation to the BbsI sites. By digesting the L0 construct with BsaI, the incorporated basic part is excised from the backbone, all the while retaining its specific set of overhangs. This makes it possible for parts occupying different cloning positions to be brought together into a cohesive Level 1 construct in a single-step reaction. This assembly of individual parts in pre-defined order is hosted by yet another plasmid backbone. In our MocloMania collection, this L1 backbone is called weird_plex and simultaneously acts as an expression vector for Leishmania tarentolae.

Please note that this part was neither designed or constructed nor altered or modified by our team. The sequence as registered is identical to the commercially available plasmid. All credit goes to the Weber / Marillonnet Labs responsible for establishing the Modular Cloning plasmid collection and depositing the respective sequences online.


level 0

size 2845 bp

antibiotic resistance spectinomycin

cloning position B3

Data

Before starting the cloning work with our self-designed and constructed L0 basic parts, we made sure that the underlying MoClo plasmid backbones were intact. We were able to confirm this by restriction digest and gel electrophoresis, see Figure 1. Please note that these plasmids were not designed or crafted by us, but simply acquired from a MoClo Toolkit that relies on the plasmids created and published by Weber et al.


  • Figure 1 | Test digest of L0 plasmid backbones using BsaI
    1 | pAGM1276 | 2244 + 598 bp
    2 | pICH41258 | 2247 + 598 bp
    3 | pAGM1287 | 2247 + 598 bp
    4 | pAGM1299 | 2247 + 598 bp
    5 | pAGM1301 | 2247 + 598 bp
    L | Thermofischer GeneRuler Plus Ladder


The MocloMania collection

This plasmid backbone is an extention to the MocloMania collection, the very first collection of genetic parts specifically designed and optimized for Modular Cloning assembly and recombinant protein expression in the protozoan parasite Leishmania tarentolae.

Are you trying to express complexly glycosylated proteins? Large antibody side chains? Human proteins that require accurate post-translational modification? Then Leishmania might be just the right organism for you! Leishmania tarentolae’s glycosylation patterns resemble those of human cells more closely than any other microbial expression host, while still delivering all the benefits of microbial production systems like easy transfection and cultivation.[2] So instead of relying on mammalian cell lines, try considering Leishmania as your new expression host of choice!

Our MocloMania collection will allow you to easily modify your protein of choice and make it suitable for downstream detection and purification procedures - all thanks to the help of Modular Cloning. This cloning system was first established by Weber et al. in 2011 and relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequence, hereby generating four nucleotide overhangs.[3] Every basic part in our collection is equipped with a specified set of overhangs that assign it to its designated position within the reading frame. These so-called cloning positions are labelled B2-B5 from upstream to downstream. By filling all positions with the basic parts of your choice, you can easily generate variable genetic constructs that code for the fusion protein of your desire.

We furthermore provide a specifically domesticated Leishmania expression vector, named weird_plex, which will package your fusion construct into a functional transcriptional unit that is optimized for high expression in Leishmania.

The best part? Because of the type IIS restriction properties and the specifity of the generated overhangs, restriction and ligation of your construct can all happen simultaneously in a simple one-step, one-pot reaction. This will safe you a lot of time and frustration in your cloning endeavours!

Do we have your attention? In the table below you can find some basic information on how our cloning system, along with most other MoClo systems, is set up. Please feel free to check out our wiki to find more information on Leishmania and Modular Cloning as well as to understand how the part that you are looking at integrates into our part collection. See you there!


MOCLO | Important nomenclature and parameters
Level What does this level contain? antibiotic resistance Enzyme used for ligation
L0 The foundation to every MoClo construct which are basic genetic units, such as coding sequences, promoters, terminators spectinomycin BbsI
L1 Several L0 parts assembled into a functional transcriptional unit, e.g. consisting of promoter, coding region and terminator ampicillin BsaI
L2 Multiple transcriptional units added into one multi-gene construct, e.g. a protein of interest fused to a resistance cassette kanamycin BbsI


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2477
    Illegal XbaI site found at 2450
    Illegal PstI site found at 2438
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2477
    Illegal PstI site found at 2438
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2477
    Illegal BamHI site found at 2456
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2477
    Illegal XbaI site found at 2450
    Illegal PstI site found at 2438
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2477
    Illegal XbaI site found at 2450
    Illegal PstI site found at 2438
    Illegal NgoMIV site found at 1081
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2225
    Illegal BsaI.rc site found at 2835
    Illegal SapI.rc site found at 2162
    Illegal SapI site found at 4


Reference Literature

  1. 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. https://doi.org/10.1371/journal.pone.0016765
  2. Langer T, Corvey C, Kroll K, Boscheinen O, Wendrich T, Dittrich W. Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae. Prep Biochem Biotechnol. 2017 Nov 26;47(10):1008-1015. doi: 10.1080/10826068.2017.1365252. Epub 2017 Aug 31. PMID: 28857681.
  3. 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. https://doi.org/10.1371/journal.pone.0016765
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