Part:BBa_K3781011

SARS-CoV-2 spike protein receptor binding domain, MocloMania B3

This part codes for the receptor binding domain of the SARS-CoV-2 spike protein. These spike proteins are part of multimeric protein structures called spikes that project outwards from the virus envelope’s outer surface. As one of the four viral structural proteins it is essential for the recognition, binding and entry of the virus into the host’s cells. This is mediated by the receptor binding domain, located in the S1 region of the spike protein, due to its specific binding to the angiotensin-converting enzyme 2 (ACE2) located on the outer membrane of many mammalian and bird cells. This binding results in a TMPRSS2 mediated proteolytic cleavage of the spike protein S2 region, exposing a viral fusion peptide and thus enabling virus entry into the host cells.

size 25.1 kDa

function coding sequence

cloning position B3

plasmid backbone pICH41258

Data

We were able to successfully clone this basic part into its respective L0 plasmid backbone and to confirm the integrity of the L0 construct via restriction digest and gel electrophoresis, see Figure 1. Furthermore, we were able to include it into a L1 construct, proving its correct adaptation towards MoClo assembly, see Figure 2.

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  Figure 1 | Test digest of L0 constructs using PstI and BstEII
  1 | L0-RBD-B3 | 2167 + 755 bp)
  2 | pICH41258 | 1850 + 995 bp)
  L | Thermofischer GeneRuler Plus Ladder [bp]
• 

  Figure 2 | Test digest of L1 constructs using SacI
  1 | pLEXSY_IE-blecherry3 | 3968 + 2515 + 1727 bp
  2 | weird_plex | 3927 + 2515 + 1268 bp
  3 | L1_sAP_RBD_mCerulean_GST | 6806 + 2515 bp
  4 | L1_sAP_RBD_mVenus_GST | 6758 + 2515 bp
  5 | L1_sAP_RBD_mCerulean_Strep8His | 6182 + 2515 bp
  6 | L1_sAP_RBD_Strep8His | 5414 + 2515 bp
  7 | L1_sAP_RBD_mVenus_Strep8His | 6134 + 2515 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]

Several L1 constructs have been assembled with this part have been successfully transfected into Leishmania and resulting tagret protein expression could be observed after purification and immunoblotting, see Figure 3. Since our project's objective was to test the efficiency of our MocloMania expression system by expressing and purifying recombinant SARS-CoV-2 receptor binding domain, this basic part was incorporated into a variety of different L1 constructs. For this, L0_RBD_B4 was usually fused to the sAP secretion tag and a C-terminal fluorescence marker as well as a terminal purification or detection tag. Most of these L1 constructs have been successfully transfected into Leishmania, resulting in recombinant protein expression that could be observed via immunostaining on western blot, see Figures 3 and 4.

• 

  Figure 3 | Immunoblot of L1 transfected Leishmania | stained against RBD
  1 | L1_sAP_RBD_mCerulean | 51.9 kDa
  2 | L1_sAP_RBD_mCerulean_Strep8His | 54.3 kDa
  3 | L1_sAP_RBD_mCerulean_GST | 78.6 kDa
  4 | L1_3xHA_RBD_mCerulean | 55.7 kDa
  n.c. | negative control | Leishmania culture
  transfected with empty L1 expression vector p.c. | RBD-GFP | 54 kDa
  L | Thermofischer PageRuler Protein Ladder [kDa]
  1. AB | ms anti-RBD
  2. AB | rb anti-ms HRP
  
• 

  Figure 4 | Immunoblot of L1 transfected Leishmania cell cultures | stained against RBD
  1 | L1_sAP_RBD_mVenus | 52.1 kDa
  2 | L1_sAP_RBD_mVenus_GST | 78.8 kDa
  3 | L1_3xHA_RBD_mVenus | 55.9 kDa
  4 | L1_sAP_RBD_HA8His | 28 kDa
  n.c. | negative control | Leishmania culture
  transfected with empty L1 expression vector
  p.c. | RBD-GFP | 52 kDa
  L | Thermofischer PageRuler Protein Ladder [kDa]
  1. AB | ms anti-RBD | 1:2,000
  2. AB | rb anti-ms HRP | 1:10,000
  

In all of the blots shown above, the constructs carrying L0_RBD_B4 were successfully stained with the help of anti-RBD antibodies. This verifies the correct recombinant expression of the underlying basic part sequence. In order to further analyse the quality of the expressed receptor binding domain, analysis blots were follwed by purification procedures to harvest produced RBD for downstream activity assays. So far, L1_sAP_RBD_mVenus_GST and L1_sAP_RBD_mCerulean_GST showed significant purification results, see Figures 5 and 6.

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  Figure 5 | Immunoblot | L1_sAP_RBD_mVenus_GST | after GST-purification
  1. AB | gt anti-GST | 1:10,000
  2. AB | rb anti-goat HRP | 1:2,000
  I Input  |  FT Flowthrough  |  W Wash  |  E Eluate  |  E.T Eluate, TCA precipitated
  L | Thermo Scientific PageRuler Protein Ladder [kDa]
  
• 

  Figure 6 | Immunoblot | L1_sAP_RBD_mCerulean_GST | after GST-purification
  1. AB | gt anti-GST | 1:10,000
  2. AB | rb anti-gt HRP | 1:2000
  I Input  |  FT Flowthrough  |  W Wash  |  E Eluate  |  E.T Eluate, TCA precipitated
  L | Thermo Scientific PageRuler Protein Ladder [kDa]
  

The MocloMania collection

This basic part is part of 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 this basic part 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

IMPORTANT

This basic part has been altered in its sequence in order to make it compatible with the iGEM RCF 1000 cloning standard.
In its original form, this part contains an endogenous SapI restriction site. This is because it was specifically designed to be utilized within the Modular Cloning system as established by Weber et al. in 2011, relying on BsaI and BbsI as the executive type IIS restriction enzymes. This MoClo standard is a very frequently used type IIS standard, especially when it comes to plant-based expression systems.

Because of its intended use within the MoClo system, this basic part is domesticated against BsaI and BbsI, but not against SapI. Upon registration of this part in the iGEM Registry, the endogenous SapI restriction site was disrupted via in silico introduction of a silent mutation. We decided to do this in order to increase the chance of the judges actually taking a look at this part page and not having the part overlooked due to incompatibility.

Since the part’s amino acid sequence remains unchanged, users interested in utilizing this part for type IIS loop assembly can rely on the sequence as listed on this page. For users interested in applying this basic part to their MoClo based cloning approaches, in silico reversion of the mutation might be beneficial, since SapI proved to be a valuable enzyme for confirming part insertion via restriction digest.

We highly recommend that researchers and iGEM teams utilize this basic part within the MocloMania cloning system, since it is codon optimized towards Leishmania tarentolae and may otherwise negatively influence expression yield in other organisms.

MUTATION | bp 634 - 636 | AGC → TCG

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 520
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 520
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 458
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 520
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 520
  Illegal NgoMIV site found at 606
  
• 1000
  COMPATIBLE WITH RFC[1000]