Part:BBa_K3711047

AOX1-α factor-ACC-AOX1 Terminator

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2440
  Illegal NheI site found at 2938
  Illegal NheI site found at 2980
  
• 21
  INCOMPATIBLE WITH RFC[21]

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

  Illegal NgoMIV site found at 1898
  Illegal NgoMIV site found at 2658
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1560
  Illegal BsaI.rc site found at 2558
  Illegal BsaI.rc site found at 2862
  

Description

This is a composite component for expressing ACC outside the cell. ACC is transcribed and translated into Acetyl CoA carboxylase，which is the key enzyme in the synthesis of lycopene. It participates in the transformation from acetyl coenzyme A to malonyl coenzyme . AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, ACC is translated and excreted from the cell.

Usage and Biology

Acetyl-CoA carboxylase (ACC) is a biotin enzyme that can catalyze the reaction of "acetyl-CoA+ATP+HCO3→malonyl-CoA+ADP+Pi". It exists widely in nature. ACC is a rate-limiting enzyme for ab initio synthesis of fatty acids, which catalyzes acetyl-CoA to malonyl-CoA, which eventually forms C16 acyl-CoA. ACC can be divided into multi-subunit ACC and multi-functional ACC. Polysubunit ACC exists in plants and bacteria and consists of four subunits, namely, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and two subunits of carboxyltransferase (CT), α-CT and β-CT. Multifunctional ACC mostly exists in eukaryotes. ACC has been used in the drug design of obesity, diabetes and plant herbicides, and is also a target gene for some crops.

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.