Part:BBa_K4032104

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

NC_000913.3 https://www.ncbi.nlm.nih.gov/gene/948088

Contents :

・The lac promoter and lacZ Part:BBa J33207 - parts.igem.org

・The native RBS

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

・The double terminator Part:BBa B0015 - parts.igem.org

Usage and Biology

This enzyme hydrolyzes of (1-4)-alpha-D-glycosidic linkages in polysaccharides containing three or more (1-4)-alpha-linked D-glucose units. See details NC_000913.3 (https://www.ncbi.nlm.nih.gov/gene/948088).

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

Design

The lac promoter and the double terminator are added to BBa_K40320xx, forming this part.

The lac promoter and the double terminator are used from BBa_J04450 (https://parts.igem.org/Part:BBa_J04450) .

This part was created by In-Fusion method using BBa_J04450 (https://parts.igem.org/Part:BBa_J04450) and BBa_K523006 (https://parts.igem.org/Part: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 of amylase･RFP

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°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.

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, 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]