Part:BBa_K4905016

mneongreen with elastin-like polypeptide

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
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This part was made to obtain a hydrophobic fluorescent protein that could be used for FRAP imaging experiments to characterize part BBa_K4905006. It consists of the fluorescent protein mNeonGreen BBa_K1761003 and a hydrophobic Elastin-Like Polypeptide sequence BBa_K4905003.

Characterization

The part was cloned into a pBAD vector as evidenced by a double digestion, with restriction enzymes cutting at both ends of the gene fragment (Figure 1).

Figure 1: Double digestion of pBad vector containing [A3G2]₁₂-mNeonGreen

Using this vector, [A3G2]₁₂-mNeonGreen was expressed in E. coli BL21(DE3). Evidence of expression can be seen in Figure 2, where the protein was loaded on an SDS-PAGE gel before and after purification with inverse transition cycling. In addition, the cell pellet showed fluorescence when excited with a blue light.

Figure 2: A) SDS-PAGE gel showing the co-expressed proteins (left three lanes) versus a negative control in which no arabinose was added and therefore no expression of [A3G2]₁₂-mNeonGreen was induced (right three lanes). B) Bacterial cell pellet excited by a blue light transilluminator showing the fluorescence emitted by mNeonGreen.