Part:BBa_K3781213

L1_sAP_RBD_Strep8His, MocloMania Composite

This composite part contains the sAP secretion signal, a coding sequence for the SARS-CoV-2 receptor binding domain as well as a C-terminal Strep8His-tag. The invidual basic parts are joined together by the MoClo connectors for B2/B3 and B4/B5.

L1_sAP_RBD_Strep8His 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 or a Strep-Tactin column, the Strep8His-tagged RBD should be able to be purified.

level 1

size 810 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 PstI
  1 | L1_sAP_RBD_Strep8His | 3894 + 1323 + 991 + 991 + 757 bp
  2 | L1_3xHA_RBD_Strep8His | 3867 + 1323 + 991 + 991 + 757 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

Despite not being able to be purified, culture supernatant of L1_sAP_RBD_Strep8His transfected culture was tested for functional activity of the included receptor binding domain in an ACE2 binding assay. For this, we used human HEK 293T +ACE2 +TMPRSS2 (hek⁺) cells that we were given to us by our sponsor VectorBuilder. This specific cell line has a mutation that renders an overexpression of the RBD receptor angiotensin I converting enzyme 2, short ACE2. As negative control we employed HEK 293T cells that don’t express ACE2 (hek^-). For a specific protocol for the conduction of this activity assay utilizing human cell culture, please refer to our Experiments page.

For the activity assay seen in Figure 7, one well of HEK-cells was incubated with the L1_sAP_RBD_Strep8His supernatant and another well with weird_plex,for both of which the cell culture supernatant was concentrated through ammonium sulfate precipitation. One further well of HEK-cells were incubated with purified L1_sAP_RBD_GST eluate. HEK^- -cells were incubated as negative controls.

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  Figure 7 | Immunoblot | stained against ACE2 and RBD.
  10% SDS-gel | 20 µL loaded per sample
  A | stained with ms α-ACE2 | 1:1000
  B | stained with ms α-RBD | 1:2000
  1 | HEK^-- cells incubated with 200 µL RBD_TEV_GST | purified
  2 | HEK⁺-cells incubated with 1.5 mL RBD_Strep8His | concentrated through ammonium sulfate precipitation
  3 | HEK⁺-cells incubated with 1.5 mL weird_plex | to rule out cross reaction with Leishmania supernatant
  4 | HEK⁺- cells incubated with 200 µL RBD_TEV_GST | purified
  5 | HEK^--cells incubated with 1.5 mL RBD_Strep8His | concentrated through ammonium sulfate precipitation
  6 | HEK⁺-cells only
  7 | HEK^-cells only
  L | Thermo Scientific PageRuler Protein Ladder
  

For the fusion protein RBD_Strep8His on HEK⁺ cells, we can see a band at the expected height (27 kDa), which is a first indication of functionality of our protein RBD. Furthermore, the band is not present in the negative control (Hek^--RBD-Strep8His) where we incubated the HEK^--cells with the same sample. This proves that the band is not a cross reaction of the antibody with any other proteins of the HEK cells or Leishmania supernatant.

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