Difference between revisions of "Part:BBa K3629025"

(→‎Usage and Biology)
(→‎References)
Line 23: Line 23:
  
 
===References===
 
===References===
 +
1.[online] https://www.fpbase.org/protein/mcherry/ (Accessed October 26, 2020)
  
 +
2.Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 05:25, 26 October 2020

mCherry expression construct with Gibson homology

mCherry expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)XPR2 terminator(BBa_K3629004), codon optimized mCherry coding sequence (BBa_K3629021) and a pair of gPCR primer binding site for Yarrowia lipolytica.

Usage and Biology

The construct is designed for optimal expression of mCherry (BBa_K3629021) in Yarrowia lipolytica.

mCherry (BBa_K3629021)is a monomeric red fluorescent protein that is derived from DsRed of Discosoma. mCherry's optimal excitation wavelength is 586nm and its optimal emission wavelength is 610nm (1). mCherry could be used to tag components and proteins within the cell which can then be visualized using fluorescence spectroscopy and microscopy.

Expression of mCherry is under the TEF intronic promoter (BBa_K3629001) which is the natural promoter found in the wild-type Yarrowia lipolytica for the Translation Elongation Factor 1 (TEF1) gene; it is a strong constitutive promoter. It also contains the first intron of the gene for stronger expression (2).

Design

The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to either the LEU4 overproducing construct (BBa_K3629023) or the TRP2 overproducing construct (BBa_K3629024)on the 5' side and to the nourseothricin resistance expression construct (BBa_K3629012) on the 3' side. The Gibson homology sequences can also be used to easily add this fluorescent protein to any construct that contains the same homology sequences.

The construct was designed to be ultimately integrated into the genome of Yarrowia lipolytica. Therefore, a pair of gPCR primer binding sites flank the construct and can be used to help determine the location of the integration of the construct in the genome.

mCherry coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1620
    Illegal NheI site found at 121
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1620
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 1000
    COMPATIBLE WITH RFC[1000]

References

1.[online] https://www.fpbase.org/protein/mcherry/ (Accessed October 26, 2020)

2.Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11