Difference between revisions of "Part:BBa K3781003"
 
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<h1>Data</h1>
 
<h1>Data</h1>
  
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We were able to <b>successfully clone</b> this basic part into its respective L0 plasmid backbone and to <b>confirm</b> the integrity of the L0 construct via <b>restriction digest</b> and gel electrophoresis, see <i>Figure 1</i>. This part has been transfected into Leishmania, but it has <b>not yet</b> been tested for expression and functionality after purification.
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We were able to <b>successfully clone</b> this basic part into its respective L0 plasmid backbone and to <b>confirm</b> the integrity of the L0 construct via <b>restriction digest</b> and gel electrophoresis, see <i>Figure 1</i>. This part has <b>not yet</b> been introduced into a <b>L1 construct</b> and thus has not yet been <b>transfected</b> into <i>Leishmania</i> to test expression and functionality. We are working hard on realizing these experiments in order to qualitatively <b>validate</b> the functionality of our basic part.
  
  
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<b>3</b> &#124; <html><a href="https://parts.igem.org/Part:BBa_K3781101">pAGM1287</a></html> &#124; 2845 bp<br>
 
<b>3</b> &#124; <html><a href="https://parts.igem.org/Part:BBa_K3781101">pAGM1287</a></html> &#124; 2845 bp<br>
 
<b>L</b> &#124; Thermofischer GeneRuler Plus Ladder [bp]]] </li>
 
<b>L</b> &#124; Thermofischer GeneRuler Plus Ladder [bp]]] </li>
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<li style="display: inline-block;"> [[File:|thumb|none|400px|<b>Figure 2 &#124; Test digest of L1 constructs</b> using <i>HindIII</i><br><b>1.</b> L1_sAP_RBD_3xHA &#124; 3792 + 3198 + 981 bp<br><b>2.</b> L1_sAP_RBD_GST &#124; 3792 + 3198 + 1563 bp<br><b>3.</b> L1_sAP_mCerulean_GST &#124; 3792 + 3198 + 1656 bp<br><b>L:</b> Thermofischer GeneRuler Plus Ladder [bp]]] </li>
 
 
</ul></div>
 
</ul></div>
  

Latest revision as of 22:58, 21 October 2021


mVenus, MoCloMania B3_B4

MocloMania

This part codes for the yellow fluorescent protein with the poetic name mVenus.[1] It is a deviation of the famous green fluorescent protein GFP originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962.[2] 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.[3]


size 26.9 kDa

function fluorescent tag

excitation wavelength 515 nm

emission wavelength 537 nm

cloning position B3_B4

plasmid backbone pAGM1287

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. This part has not yet been introduced into a L1 construct and thus has not yet been transfected into Leishmania to test expression and functionality. We are working hard on realizing these experiments in order to qualitatively validate the functionality of our basic part.


  • Figure 1 | Test digest of L0_B3_B4 parts using BstEII
    1 | L0_mCerulean_B3_B4 | 1835 + 1180 bp
    2 | L0_mVenus_B3_B4 | 1823 + 1144 bp
    3 | pAGM1287 | 2845 bp
    L | Thermofischer GeneRuler Plus Ladder [bp]


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.[4] 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.[5] 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


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]


Reference Literature

  1. ↑ Gert-Jan Kremers, Joachim Goedhart, Erik B. van Munster, and Theodorus W. J. Gadella. Cyan and Yellow Super Fluorescent Proteins with Improved Brightness, Protein Folding, and FRET Förster Radius, Biochemistry 2006 45 (21), 6570-6580, DOI: 10.1021/bi0516273
  2. ↑ Shimomura, O., Johnson, F.H. and Saiga, Y. (1962) Extraction, Purification, and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 59, 223-239. http://dx.doi.org/10.1002/jcp.1030590302
  3. ↑ M.A. Rizzo, D.W. Piston High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy Biophys. J., 88 (2005), pp. L14-L16
  4. ↑ Langer T, Corvey C, Kroll K, Boscheinen O, Wendrich T, Dittrich W. Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae. Prep Biochem Biotechnol. 2017 Nov 26;47(10):1008-1015. doi: 10.1080/10826068.2017.1365252. Epub 2017 Aug 31. PMID: 28857681.
  5. ↑ Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. https://doi.org/10.1371/journal.pone.0016765