Part:BBa_K3711075
Panb1-ACC-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1629
Illegal NheI site found at 2127
Illegal NheI site found at 2169 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 124
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1087
Illegal NgoMIV site found at 1847 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 749
Illegal BsaI.rc site found at 1747
Illegal BsaI.rc site found at 2051
Description
This is a composite part for intracellular expression of ACC. Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, ACC is expressed and participates in the production from acetyl CoA to malonyl coenzyme.
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
Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.
The bands of Panb1-CUS-AOX1 Terminator (2000+bp), Panb1-ACC-AOX1 Terminator (3000bp), Panb1-4CL-AOX1 Terminator (2500+bp) and Panb1-crtI-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 2158bp, 2832bp, 2688bp and 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
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