Part:BBa_K4032104

lacI+lacZ+RBS+amylase-RFP+double terminator

This part contains sequences of fuse protein of amylase and RFP.

This part is the addition of a lac promoter and double terminator to BBa_K40320xx. Lac promoter and double terminator were used as contained in BBa_J04450.

This enzyme hydrolyzes of (1-4)-alpha-D-glycosidic linkages in polysaccharides containing three or more (1-4)-alpha-linked D-glucose units. Red fluorescence of RFP is observed under the fluorescence microscope.

This part was created by In-Fusion method using BBa_J04450 as inverse and BBa_K523006 as insert.

Fig. 1 method

Experiments

【image】

Fig. 2　The growth of E.coli (DH5α) expressing of amylase･RFP

Pre-culture was performed at 37°C for 9 hours. Incubation was carried out at 37°C, 130 rpm. Four hours after the start of incubation, 0.5 mM IPTG was added to the amylase･RFP culture solution when OD = 0.633. We measured OD600 every approximately 4 hours.

SDS To investigate the expression of amylase・RFP from E. coli and coral, bacteria transformed with BL21 (DE3) were cultured in an LB medium containing chloramphenicol. After incubation of E. coli at 37°C and 130 rpm for 16 hours, the cells were inoculated into a new medium and cultured in liquid until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and E. coli was cultured overnight for 10 hours at 25°C and 130 rpm. The cultured bacteria were sonicated using Sonication buffer (Phosphate buffer solution (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

【image】

Figure ??. SDS-PAGE gel for quantification of amylase-RFP. M, molecular mass markers; WT, wild type; amy-RFP, amylase-RFP from E.coli and coral; P, pellet; S, solubility.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1841
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 607
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 977
  Illegal AgeI site found at 2142
  Illegal AgeI site found at 3221
  Illegal AgeI site found at 3333
  
• 1000
  COMPATIBLE WITH RFC[1000]