Part:BBa_K1717171

Protein coding sequence for KeratinaseA

Keratinases are proteolytic enzymes which break the disulphide bonds in various keratin substrates. KERA is a basic part comprised from a synthesized KERA protein coding sequence, optimized for expression in E. coli and most active in degrading keratin in feathers. This gene, when expressed in cells, can be tested for activity through looking for zones of clearing on skim milk agar plates where cells are growing, as well as by looking at degradation of keratin-containing substrates (feathers, hair, chemically synthesized keratin, etc.) when grown with cells.

SZ_SHD 2021's Improvement

Introduction

The composite part BBa_K1717171 is an optimized version of KERA that can be constructed into pSB1C3 plasmid with the highest protein expression in E.coli BL21(DE3). The KERA is a basic part comprised from a synthesized KERA protein coding sequence. The gene can be expressed in cells by looking for clear areas of cell growth on AGAR plates of skimmed milk, and by observing keratin-containing substrates (feathers, hair, chemosynthetic keratin, etc.) growing with cells. Detailed optimization data can be checked here: https://2021.igem.org/wiki/images/0/04/T--SZ_SHD--dataset.csv

Plasmid Construction

Figure 1. The construction of optimized KERA on pSB1C3 backbone, with 6xHis-tag for purification. The combination of promoter, RBS, and terminator is with the best performance in expression.

Experiment details

• Throughput: 2500
• Organism: E. coli
• Strain type: BL21(DE3)
• Temperature: 37°C
• Media: 250 ml LB
• Period of time: 16 hours
• Plasmid backbone: pSB1C3

A. Vec platform was used to test the expression of KERA in more than 2000 random expression vectors. Different regulatory elements used in the vector can result in nearly 10 orders of magnitude difference in KERA expression.

Figure 2. The data plot of KERA optimization. Within more than 2000 protein yields, the final relative unit of the expression vector is about 10 orders of magnitude higher than before.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 130
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 391
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 761
  Illegal SapI site found at 1064