Part:BBa_K3895016

Optimized keratinase KerA

Introduction

This composite part 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 (BBa_K1717171) 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 326
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 587
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1369
  Illegal SapI site found at 957
  Illegal SapI site found at 1260