Part:BBa_K3711045

AOX1-α factor-LOX2-AOX1 Terminator

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2428
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1526
  Illegal XhoI site found at 1187
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2042
  Illegal AgeI site found at 2719
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2508
  

Description

This is a composite component for expressing LOX2 outside the cell. LOX2 is transcribed and translated into Lipoxygenase 2，which is the key enzyme for degradation of curcumin. It participates in the transformation from Polyunsaturated fatty acid to hydroperoxides (OOHs) ,then the resulting peroxides immediately react with lycopene to promote the degradation of lycopene. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, LOX2 is translated and excreted from the cell.

Usage and Biology

Lipoxygenase (LOX) attaches oxygen to the acyl groups of polyunsaturated fatty acids or glycerides to form corresponding hydroperoxides. Lipoxygenase and peroxidase are involved in the degradation of lycopene in food, which requires the existence of oxygen and activating cofactors at the same time. Because lycopene is a compound of carotenoids, the biological function of β-carotene in carotenoids is attributed to its ability to scavenge free radicals and physical quenching of singlet oxygen. And produce vitamin A (retinol). Although lycopene can not be converted into vitamin A, it has a strong effect of scavenging singlet oxygen, scavenging free radicals and inhibiting lipid peroxidation. Lycopene has the strongest antioxidant effect among carotenoids, especially at twice the rate of β-carotene. In the process of lycopene degradation caused by lipoxygenase, lipoxygenase first catalyzes the oxidation of unsaturated or polyunsaturated fatty acids, and the resulting peroxide reacts with lycopene to promote the degradation of lycopene, in order to prevent the rancidity of oily food.

Molecular cloning

Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.

Figure1: Colony PCR results of AOX1-α factor-CUS-AOX1 Terminator, AOX1-α factor-ACC-AOX1 Terminator, AOX1-α factor-4CL-AOX1 Terminator and AOX1-α factor-LOX2-AOX1 Terminator transformed E.coli.

The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator (3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from colony PCR are identical to the theoretical lengths of 3046bp, 3619bp, 3523bp and 4528bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Figure2:Colony PCR result of yeast after electroporation through electrophoresis.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.

Figure3:SDS-PAGE result of Laccase GS115 4CL LOX2 ACC detecetion in the supernatant.

Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa)but still within explainable and acceptable range, which could be evidence of successful expression.

Enzyme activity determination

For LOX2: use 27ul linoleic acid, 25ul tween 20, 8ml ddH20, 3M/L NaOH 183ul, and dilute to 50ml as a solution; take 200ul substrate, 1ml supernatant, and 1ml pH6.0 buffer for measuring absorbance changes at 234nm. And every 0.001 increase in absorbance is regarded as an enzyme activity unit. Its activity was successfully detected.