Part:BBa_K3781012

mCerulean, MocloMania B4

This part codes for the cyan fluorescent protein mCerulean. The protein was originally derived from the green fluorescent protein GFP which was first isolated from the bioluminescent jellyfish Aequorea victoria in 1962. It can be fused to a protein of interest, making it accessible to easy and non-invasive screening opportunities that involve fluorescence monitoring, such as fluorescence spectroscopy or microscopy.

size 26.8 kDa

function fluorescent tag

excitation wavelength 435 nm

emission wavelength 477 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 different L1 constructs assembled with L0_mCerulean_B4 have been successfully transfected into Leishmania, resulting in recombinant protein expression that could be observed after immunostaining on western blot.

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  Figure 3 | Immunoblot of L1 transfected Leishmania cell cultures | 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
  

Looking at Figure 3, we see that both constructs that employ L0_mCerulean_B4, namely 2 | L1_sAP_RBD_mCerulean_Strep8His and 3 | L1_sAP_RBD_mCerulean_GST, show definite protein bands when stained against the SARS-CoV-2 receptor binding domain. As both constructs contain the sAP secretion tag, we can see that protein expression is detected both in the cell lysate P+ as well as in the culture supernatant S+. In silico calculations predict construct 2 to weigh about 50 kDa and construct 3 around 80 kDa. Taking into consideration small deficiencies in gel density due to high percentage of acrylamide, both constructs' upper bands can be attributed to that respective size. However, a smaller band, running at around 28 kDa can be observed for both constructs, but exclusively in the cell culture supernatant. This leads to the hypothesis of extracellular cleavage processes occurring 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_mCerulean_B4, so far L1_sAP_RBD_mCerulean_GST has been successfully purified. Even tho this purification didn't yield substantial amounts of intact fusion protein, we still tried to investigate the activity of the purified RBD by incubating human HEK cell lines overexpressing the ACE2 recpetor with the RBD eluate and observing fluorescence via fluorescence microscopy, see Figure 4. The overexpressing cell line is abbreviated as HEK⁺. As a negative control we used HEK 293T cells that don’t express ACE2, HEK^-. For both cells, the nucleus was visualized with the help of DAPI. Additionally, the cells were treated with a solution that extends the emission/extinction of the fluorophores, ProLong^™ Glass Antifade Mountant.

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  Figure 4 | Fluorescence microscopy HEK-cells transfected with L1_sAP_RBD_mCerulean_GST
  A-C | HEK^--cells | DAPI nucleus stain | incubation with RBD_mCerulean_TEV_GST
  D-F | HEK⁺-cells | DAPI nucleus stain | incubation with RBD_mCerulean_TEV_GST
  emission | 435 nm
  extinction | 477 nm
  

Due to the visible disintegration of the nucleus in many of the HEK⁺ cells, it is evident that they are mutants and therefore not as fit as the HEK^-- cells. The mCerulean fluorophore has the same excitation wavelength as DAPI, which makes it difficult to distinguish them from one another. Nevertheless, a slight shimmer around the HEK⁺ cells is visible which is not apparent in the HEK^- cells. That could indicate the binding of our RBD_mCerulean fusion protein to the ACE2 receptors on the cells' outer membrane. Having mCerulean and DAPI fluorescence spectra overlapping is an unanticipated issue which we plan to resolve by introducing additional fluorescent tags into our library, covering a broader range of excitation and emission wavelengths.

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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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