Part:BBa_K3781206

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 cause 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 822 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.

• 

  Figure 1 | Test digest of L1 constructs using SacI
  1 | weird_plex | 3927 + 2515 + 1268 bp
  2 | L1_sAP_RBD_HA8His | 5423 + 2515 bp
  3 | L1_sAP_Strep_mCerulean_GST | 6149 + 2515 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]

This L1 composite part has been successfully transfected into Leishmania and recombinant protein expression could be observed via immunostaining on western blot, see Figure 2.

• 

  Figure 2 | Immunoblot of L1 transfected Leishmania | stained against His

  1 | L1_sAP_RBD_HA8His | 28 kDa
  2 | L1_sAP_RBD_Strep8His | 27.7 kDa
  p.c. | exemplary His-tagged protein | 23 kDa
  n.c. | culture transfected with empty L1 expression vector
  L | Thermo Scientific PageRuler Protein Ladder [kDa]
  1. AB | ms anti-His | 1:4,000
  2. AB | rb anti-ms HRP | 1:10,000

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. 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. 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 selection marker	kanamycin	BbsI

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 598
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 598
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 536
  Illegal XhoI site found at 6
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 598
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 598
  Illegal NgoMIV site found at 684
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 708