Plasmid

Part:BBa_K4427008

Designed by: Chanjuan Jin   Group: iGEM22_JLU_China   (2022-09-30)


Pet-30a-KdcA 

Plasmid containing KdcA

part-008.png

To initially determine whether the engineered bacteria expressed KDCA, we performed SDS-Page of the proteins in the pre-induction broth, the post-induction broth, the supernatant and the precipitate after induction of the crude extract. the results showed that the target protein KDCA was present in all four samples simultaneously, but was lower in the non-IPTG induced samples and more of the other proteins were present. The supernatant after induction of the crude extract had a large amount of the target protein we needed.

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（1) UV absorption spectrum of coenzyme NADH in the KDCA cascade reaction We used pyruvic acid as the reaction substrate and a citric acid/sodium hydroxide solution at pH=6 as a buffer, KDCA can catalyse the reaction of pyruvic acid to form acetaldehyde, and then use ADH1 to catalyse the reaction of acetaldehyde to ethanol to determine the activity of KDCA. We used a UV spectrophotometer to measure the absorbance of the solution at reaction times of 1, 2, 3, 4 and 5 min. The results showed that NADH had a high absorbance when irradiated with UV light at a wavelength of 340nm and its content decreased with increasing reaction time, proving that our enzyme was active.

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(2) Concentration-dependent enzymatic activity of KDCA Following the determination of the absorbance spectra of KDCA coenzyme, we determined that the enzyme concentration-dependent enzymatic activity of KDCA was determined using UV light at a wavelength of 340 nm. We added 10, 15, 20, 30, 35 and 40 ul of KDCA to the substrate and carried out six control experiments. The results of the experiments showed that the rate of substrate reduction was increasingly faster with increasing enzyme concentration at enzyme solution volumes of 10-30ul, i.e. the higher the enzyme activity. The reaction rate at 35 ul of enzyme solution slowed down compared to 30 ul, but the rate increased again at 40 ul.

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Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 5034
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 6723
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 5034
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 5034
    Illegal NgoMIV site found at 137
    Illegal NgoMIV site found at 3184
    Illegal NgoMIV site found at 3344
    Illegal NgoMIV site found at 4932
  • 1000
    COMPATIBLE WITH RFC[1000]


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