Difference between revisions of "Part:BBa K1033914"

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In order to measure absorbance and fluorescence spectra of amajLime we transformed the construct (consisting of J23110 Promotor, B0034 RBS and  amajLime coding sequence) into ''E. coli'' (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to this [https://2019.igem.org/Team:Humboldt_Berlin/Experiments protocol.]  
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In order to measure absorbance and fluorescence spectra of amajLime we transformed the construct (consisting of <partinfo>BBa_J23110</partinfo> Promotor, <partinfo>BBa_B0034</partinfo> RBS and  amajLime coding sequence) into ''E. coli'' (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to this [https://2019.igem.org/Team:Humboldt_Berlin/Experiments protocol.]  
  
 
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Revision as of 15:33, 16 October 2019

amajLime, yellow-green chromoprotein (incl RBS & J23110)

This chromoprotein from the coral Anemonia majano, amajLime (also known as amajCFP or amFP486), naturally exhibits strong color when expressed. The protein has an absorption maximum at 458 nm giving it a yellow-green color visible to the naked eye. Compared to many other chromoproteins, such as amilCP (BBa_K592009), amilGFP (BBa_K592010), spisPink (BBa_K1033932), asPink (BBa_K1033933) and aeBlue (BBa_K864401), the color development is slower. The color is readily observed in both LB or on agar plates after 24-48 hours of incubation. The protein amajLime has significant sequence homologies with proteins in the GFP family.

Characterisation

Team: Humboldt_Berlin 2019

ExPASy ProtParam Results:


Number of amino acids: 228

Molecular weight: 25185.49 Da

Theoretical pI: 7.27

Total number of negatively charged residues (Asp + Glu): 22

Total number of positively charged residues (Arg + Lys): 22


Extinction coefficients:

Ext. coefficient 26150 M-1 cm-1

Abs 0.1% (=1 g/l) 1.038, assuming all pairs of Cys residues form cystines

Ext. coefficient 25900 M-1 cm-1

Abs 0.1% (=1 g/l) 1.028, assuming all Cys residues are reduced

Instability index: The instability index (II) is computed to be 27.07. This classifies the protein as stable.

Aliphatic index: 51.71

Grand average of hydropathicity (GRAVY): -0.379


In order to measure absorbance and fluorescence spectra of amajLime we transformed the construct (consisting of BBa_J23110 Promotor, BBa_B0034 RBS and amajLime coding sequence) into E. coli (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to this protocol.

  • Liquid culture of E. coli expressing amajLime

  • Cell pellet after centrifugation

  • Cleared cell lysate

The fluorescence and absorbance spectra was measured for 24 samples of 150 µl lysate in 96-well plate on TECAN Plate Reader Infinite 200 Pro. In figure 1 you can see the relative absorbance and emission spectra including the respective standard deviation.


Table 1. Parameters utilized for absorbance and fluorescence spectra
Parameter Value
Number of samples 24
Absorbance Scan: Excitation Wavelength measurement range (nm) [300-800]
Fluorescence Scan: Emission Wavelength measurement range (nm) [475-550]
Fluorescence Scan: Excitation wavelength (nm) 445
Gain (G) 52
Number of Flashes 25
Integration Time (µs) 20
Lag Time (µs) 0
Settle Time (ms) 0



Figure 1. amajLime absorbance and fluorescence spectra

References

[http://www.nature.com/nbt/journal/v17/n10/full/nbt1099_969.html] Matz, Mikhail V., et al. "Fluorescent proteins from nonbioluminescent Anthozoa species." Nature biotechnology 17.10 (1999): 969-973.

[http://www.pnas.org/content/102/36/12712.short] Henderson, J. Nathan, and S. James Remington. "Crystal structures and mutational analysis of amFP486, a cyan fluorescent protein from Anemonia majano." Proceedings of the National Academy of Sciences of the United States of America 102.36 (2005): 12712-12717.

[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.

[1] Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12:8. 10 May. 2018, doi:10.1186/s13036-018-0100-0


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]