Part:BBa_K5048065

SUMO-HSP60-FLAG

Codes for HSP60, a membrane protein of small intestinal epithelial cells, which can adhere to LAP. Codon optimized for expression in E.coli, and a FLAG tag was added for convenient detection. On the N-terminus, a Ubiquitin-like protein SMT3(SUMO)tag was added to improve the solubility of the protein.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 215
  Illegal PstI site found at 4
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 215
  Illegal PstI site found at 4
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 215
  Illegal BglII site found at 104
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 215
  Illegal PstI site found at 4
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 215
  Illegal PstI site found at 4
  Illegal NgoMIV site found at 619
  Illegal AgeI site found at 774
  Illegal AgeI site found at 1252
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

SUMO: Ubiquitin-like protein SMT3. A protein tag with a molecular weight of 11.3 kDa, is used to enhance protein solubility.
We build up this gene circuit to obtain more protein HSP60 in the supernatant, aiming to acquire more raw material for Co-IP(Fig 1).

Fig 1 The genetic circuits for SUMO-HSP60.

Source

SMT3 SUMO family protein SMT3 [ Saccharomyces cerevisiae S288C ] Gene ID: 852122

Experiments

This part acts as a system that provides more protein HSP60 in the supernatant.

The overall experiments we conducted on BBa_K5048065 include the following:

1. Verification of PCR products;
2. Verification of transformation of the plasmid.

Results

Verification of PCR products

For the genetic circuit SUMO-HSP60, DNA segments, including SUMO and HSP-pET-backbone, were amplified successfully (Fig 2).

Fig 2 The construction of SUMO-HSP60
A. The agarose gel electrophoresis of SUMO segment for SUMO-HSP60. SUMO: 325bp.
B. The agarose gel electrophoresis of HSP60-pET-backbone segment for SUMO-HSP60. HSP60-pET-backbone: 6982bp.

Verification of transformation of the plasmid

Furthermore, we connected the two DNA segments and built up plasmid SUMO-HSP60 in E. coli BL21(DE3) with single colonies containing the target band (Fig 3).

Fig 3 SUMO-HSP60 successfully transferred into E. coli BL21(DE3)
A. The plate of E. coli DH5α harboring the transferred plasmid SUMO-HSP60, which contains abundant single colonies. Resistance: Kanamycin (Kan).
B. The colony PCR for E. coli BL21(DE3). SUMO-HSP60 1 and 3-5 all have segments corresponding to the estimated DNA length. SUMO-HSP60: 2055bp
C. The sequencing result for SUMO-HSP60 in E. coli BL21(DE3), demonstrates the successful transformation.

Applications of BBa_K5048065

This part acts as a system that provides more protein HSP60 in the supernatant to achieve better results for Co-IP.