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<div style="text-align:justify;"> Figure 8: DIC and Fluorescence image of RFP expression in bacteria after induction with IPTG for 4 hrs.
 
<div style="text-align:justify;"> Figure 8: DIC and Fluorescence image of RFP expression in bacteria after induction with IPTG for 4 hrs.

Revision as of 03:38, 22 October 2019


pT7+RFP+TAL

Tyrosine Ammonia Lyase with RFP under T7 promoter

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 797
    Illegal NgoMIV site found at 1629
    Illegal AgeI site found at 593
    Illegal AgeI site found at 705
    Illegal AgeI site found at 892
    Illegal AgeI site found at 1058
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2087


Characterization of RFP and TAL (2019 SNU_India)

Following the cloning, we checked for the expression of both RFP and p-coumaric acid in bacteria. RFP was visualized by fluorescence microscopy following IPTG (0.5mM) induction for 4 hrs (Figure 8) and p-coumaric acid (Figure 9a-d) production was probed in filtered culture supernatant by mass spectrometry (Agilent Technologies 6540 UHD-Accurate Mass Q-TOF LC/MS in negative mode using methanol as solvent)

Figure 8: DIC and Fluorescence image of RFP expression in bacteria after induction with IPTG for 4 hrs.

Mass spectra of 1) Pure p-coumaric acid in methanol and 2) mass spectra of Induced culture supernatant

Reference: Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012), "Fiji: an open-source platform for biological-image analysis", Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019