Plasmid

Part:BBa_K4427007

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


Pet-22b-Adh1 

Plasmid containing adh1

As for pET-22b-Adh1.dna, we designed the lactose promoter in this plasmid, as well as inserting the protease gene for Adh1, and the AmpR resistance gene for identification of plasmid transformation.

Adh1 (alcohol dehydrogenase) converts the above-mentioned PAD catalyzed by KdcA into the final product 2-PE, a geotactic process that uses the genetically catalyzed product PAD of the former enzyme, which is then converted to the final product 2-PE by Adh1.

part-007-small.png 

SDS-PAGE:

part-007-1.png


(1) ADH1 coenzyme NADH absorption spectrum We used acetaldehyde and NADH as reaction substrates and a solution of glycine/sodium hydroxide at pH=10 as a buffer to verify the catalytic effect of ADH1 on this reaction. We used a UV spectrophotometer to measure the absorbance of the solution at reaction times of 0, 3, 6, 9, 12, 15 and 18 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.

part-007-2.png

(2)Following the determination of the absorbance spectra of ADH1 coenzyme, we determined that the enzyme concentration dependent enzymatic activity of ADH1 was measured using UV light at a wavelength of 340 nm. We added 3, 4, 4.5, 5 and 6 ul of ADH1 to the substrate and performed five sets of controlled experiments. The results of the experiments showed that the rate of substrate reduction became increasingly rapid with increasing enzyme concentration at 3-6 ul of enzyme solution, i.e. the higher the enzyme activity.

part-007-3.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1251
    Illegal PstI site found at 5274
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 5274
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1317
    Illegal XhoI site found at 158
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1251
    Illegal PstI site found at 5274
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1251
    Illegal PstI site found at 5274
    Illegal NgoMIV site found at 1349
    Illegal NgoMIV site found at 2937
    Illegal NgoMIV site found at 3097
    Illegal NgoMIV site found at 6277
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5100
    Illegal SapI site found at 4017


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