Part:BBa_K3077000

Expression of malS under lac promoter

The part BBa_K3077000 is a composite part of the periplasmic α-amylase gene encoded as malS in the E. coli. The genomic sequence of the malS gene is referenced from BBa_K1418020 registered by Team Utah State in 2014. Team Utah State removed the native ribosome binding site and the predicted cleavage signal peptide of BBa_K523001 in 2014, forming BBa_K1418020. Our teammates further designed it to be composite part by adding lac promoter BBa_R0010, strong RBS BBa_B0034, 10x-Histidine tag BBa_K844000 and double terminator BBa_B0015.

• R0010 is a conditional promoter that promotes transcription in the absence of LacI protein and CAP protein, which can be maintained by the presence of IPTG and low glucose level. This can prevent overexpression of the cell and keep it healthy.
• B0034, a strong RBS, to achieve a higher expression level.
• K844000, a 10X His-tag, to allow protein purification, is fused at the N terminus of the lipase gene.
• B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.

The gene BBa_K1418020 is the Periplasmic alpha-amylase encoded as malS in E. coli. malS is a hydrolase responsible for degrading amylose and long maltodextrins with at least 3 glucose residues, including starch[1]. It recognizes substrates from the non-reducing end and hydrolyses the α-1,4 glycosidic linkages between each glucose monomer, yielding maltotriose, maltose, and glucose with even numbers of glucose residues[1]. MalS is thought to provide a growth advantage by degrading periplasmic maltodextrins between 7 and 15 glucose units in length[1]. Since only maltooligosaccharides up to a chain length of 6 glucose units are actively transported through the cytoplasmic membrane via the membrane-bound complex of three proteins, MalF, MalG, and MalK, longer maltooligosaccharides must first be degraded by the MalS protein[1].

We failed to order the composite part BBa_K3077000 from IDT, so related assays can't be performed to test the effectiveness of the composite part.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1426
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 562
  Illegal AgeI site found at 1727
  
• 1000
  COMPATIBLE WITH RFC[1000]