Part:BBa_K4032104

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

Contents :

・The lac promoter and lacZ (from BBa_J33207)

・The native RBS (from BBa_K523001)

・The gene codes for the amylase-RFP fusion protein.（BBa_K4032003）

・The double terminator (from BBa_B0015)

See NCBI: NC_000913.3for information on the amylase gene malS.

Usage and Biology

This enzyme hydrolyzes of (1-4)-α-D-glycosidic linkages in polysaccharides containing three or more (1-4)-α-linked D-glucose units. See NCBI: NC_000913.3for details.

In the case of this part, Red red fluorescence of RFP is observed with a fluorescence microscope, but isn’t observed under the natural light source.

Design

The lac promoter and the double terminator are added to BBa_K4032006, forming this part.

The lac promoter and the double terminator are from BBa_J04450.

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

Fig. 1　The plasmid design of BBa_K4032104

Experiments

Time course

Fig. 2　The growth of E. coli (DH5α) expressing amylase-RFP fusion protein

Pre-culture : 37 ℃,9 h (130 rpm)

Culture : 37 ℃ (130rpm)

･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63)

･Measuring OD600 every approx. 4 hours

SDS-PAGE

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 ℃ 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 ℃ 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.

Fig. 3 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, soluble.

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]