Part:BBa_K4221003

EGFP

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 653
  

Usage

Aqueous two-phase separation (ATPS) is a liquid-liquid fractionation technique effectively used for protein separation and purification[1]. When a protein fuses with a hydrophobin, the hydrophobin changes the hydrophobicity of the protein, which causes the protein to aggregate into the surfactants.

Our team is trying to improve traditional ATPS by incorporating a continuous-flow system and replacing fungal hydrophobins with BslA. Using EGFP[2] as target protein can visually observe fluorescent protein (EGFP,target protein) showing green fluorescence in the process of protein expression and two-phase extraction, so as to determine the separation and purification effect.

Biology

Green fluorescent protein (GFP)[3] was originally identified from the jellyfish (Aequorea victoria). When the jellyfish luminescent protein binds Ca2+, it emits blue fluorescence, which is further excited to produce green fluorescence，in order to improve the detection sensitivity of Reporter gene, Guohong et al. replaced Ser65 with Thr and Phe64 with Leu, which increased the fluorescence intensity of EGFP by 35 times compared with GFP.

Design Consideration

The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli and Rosetta E. coli.

The construction includes:

EGFP is fused with BslA with a GS linker（GGTGGTGGCGGCAGCGGCGGAGGCGGTAGT） and TEVlinker（GAAAACCTGTACTTCCAGGGTTCTGGT）

Reference

[1] E Mustalahti, M Saloheimo, J J. JoensuuIntracellular protein production in Trichodermareesei (Hypocreajecorina) with hydrophobin fusion technology[J]. New Biotechnology, 2013(30)

[2]Aijia J, Xibin N. Construction and Expression of Prokaryotic Expression Vector pET28a-EGFP[J]. JOURNAL OF MICROBIOLOGY, 2011, 31(4):69-73.

[3]J.Ma Green fluorescent protein[J](in Chinese). Chemistry, 2009(3):243-250.