Part:BBa_K3781014

mVenus, MocloMania B4

This part codes for the yellow fluorescent protein with the poetic name mVenus. It is a deviation of the famous green fluorescent protein GFP originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962. By coupling a protein of interest with a fluorescent tag such as mVenus, the target protein can be easily and non-invasively detected within living cells as well as cell preparations with the help of fluorescence spectroscopy and microscopy.

size 26.9 kDa

function fluorescent tag

excitation wavelength 515 nm

emission wavelength 537 nm

cloning position B4

plasmid backbone pAGM1299

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 B4 parts using BsaI
  1 | pAGM1299 | 2247 + 598 bp
  2 | L0_RBD_B4 | 2247 + 675 bp
  3 | L0_mCerulean_B4 | 2247 + 720 bp
  4 | L0_mVenus_B4 | 2247 + 720 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 assembled with L0_mVenus_B4 have been successfully transfected into Leishmania, yet resulting recombinant protein expression could only be observed on immunoblots for L1_sAP_RBD_mVenus_GST, see Figure 3.

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  Figure 3 | 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
  

Looking at Figure 3, we see that construct 2 | L1_sAP_RBD_mVenus_GST shows definite protein bands in the cell culture supernatant, S+, when stained against the SARS-CoV-2 receptor binding domain. As it contains the sAP secretion tag, protein expression is expected to be prevalent in the exterior culture medium S+. In silico calculation predicts construct 2 to weigh around 80 kDa. This is contradictory to the running hight of the bands, the band attributed to the full-length protein appearing over 100 kDa in size. Since the band pattern closely resembles the one seen in other fluorophore carrying constructs, e.g. L1_sAP_RBD_mCerulean_GST, we assume that this size shift is caused by technical deficiencies of the SDS-PAGE conduct. Shift of bands to unexplicably higher sizes might stem from a problem in gel density, caused for example by excess acrylamide concentrations.

Taking into consideration the assumed deficiencies in gel density, the construct's upper band can be attributed to the full-length fusion protein. However, a smaller band, running at 30 kDa (assumed to be at ca. 27 kDa) can be observed. Since this smaller band is also detected with anti-RBD antibodies, it is hypothesized that extracellular cleavage processes separate the RBD from its fusion tags after secretion of the fusion protein into the culture medium. For more information on this, please consult the sAP secretion tag part site.

Out of all the L1 constructs employing L0_mVenus_B4, so far L1_sAP_RBD_mVenus_GST has been successfully purified. This purification didn't yield sufficient amounts of intact fusion protein for downstream analysis, because we are struggling with premature cleavage of our GST fusion tag. Therefore, fluorescence spectroscopy of purified protein samples has not yet been conducted, neither has fluorescence microscopy of Leishmania producing recombinant mVenus. Thus, at this point in time, functionality of L0_mVenus_B4 protein cannot be definitively verified. We are working hard on optimizing expression and purification yields in order to finalize part validation and implementation towards their respective functionality.

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 resistance cassette	kanamycin	BbsI

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 202
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 202
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 202
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 202
  Illegal NgoMIV site found at 679
  
• 1000
  COMPATIBLE WITH RFC[1000]