Revision as of 12:23, 16 October 2021

L1_sAP_RBD_HA8His, MocloMania Composite

This composite part contains the sAP secretion signal, a coding sequence for the SARS-CoV-2 receptor binding domain and a C-terminal HA8His-tag. The invidual basic parts are joined together by the MoClo connectors for B2/B3 and B4/B5.

The utilized Ha8His-tag in cloning position B5 is not part of the MocloMania collection, but has instead been adapted from our supervising working group. The iGEM Team TU_Kaiserslautern 2019 has already registered and characterized this basic part within their project, a MoClo parts library adapted towards expression in the green algae Chlamydomonas reinhardtii. It was utilized by us because, at that time, none of our MocloMania B5 parts were cloned successfully, but we were in need of a functional part to occupy the B5 cloning position. Due to the high similarity in codon usage between Chlamydomonas and Leishmania, transfer of the part didn't cost any additional cloning work.

L1_sAP_RBD_HA8His allows for transgenic expression of the SARS-CoV2 receptor binding domain in Leishmania tarentolae and secretion of the fusion protein into the culture medium. By concentrating the culture medium and running it over a nickel/cobalt resin column, the HA8His-tagged fusion protein should be able to be purified. Furthermore, the HA allows for detection of recombinant protein via immunostaining on western blot.

level 1

size 1530 bp

antibiotic resistance in E. Coli ampicillin

plasmid backbone weird_plex

Data

We were able to successfully assemble this composite part into our L1 expression vector weird_plex and confirm the integrity of the resulting L1 construct via restriction digest and gel electrophoresis, see Figure 1. Along with subsequent sequencing, this verified the correct adaptation of the underlying basic parts to the MoClo cloning standard. This composite part has been transfected into Leishmania, but it has not yet been tested for expression and purification functionality.

The MocloMania collection

This L1 construct was assembled using basic parts from 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.^[1] 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.^[2] 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!

1. ↑ 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.
2. ↑ 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