Part:BBa_K4940000

GLF-his

GLF encodes a glucose facilitator, located on the cell membrane of Escherichia coli, which can enhance glucose utilization.

Usage and Biology

It has the activity of mediating glucose diffusion, transporting unphosphorylated glucose into the cell without the need to consume metabolic energy in the form of proton potential or phosphoenolpyruvate (1). The expression of this gene can increase the glucose utilization of Escherichia coli. It can also be used in the field of aromatic synthesis to increase metabolic flow, accelerate the transport of substrates (such as glucose) into the cell, and increase the supply of precursor substances (2). In our project, we constructed an NADH regenerative system, and GLF is a major component here to enhance glucose uptake.

Plasmid design and construction

GLF gene was inserted into the vector pQE-80L-Kan, and this plasmid was then transformed into E. coli BL21 star (DE3).
Figure 1. The construction of pQE-80L-Kan GLF plasmid.

Cultivation, Purification and SDS-PAGE

The recombinant E.coli was cultured and induced by IPTG to allow GLF protein expression. Since the protein was fused to a his tag, we purified the protein via affinity chromatography. The protein samples were tested by SDS-PAGE. We have already confirmed the expression of GLF based on the corresponding bands.

Figure 2. Transformation results of three plasmids in the LB agar plate. Three plasmids were all successfully transformed into bacteria, indicated by the colonies on the agar plate. (A) E. coli BL21 star (DE3) with pQE-80L-Kan glf; (B) E. coli BL21 star (DE3) with pCDFDUET-1 p450bm-3qm glcdh-II; (C) E. coli BL21 star (DE3) pET-28a prα-pol.
Figure 3. The results of the SDS-PAGE. The corresponding protein bands were indicated by the red box.

References

1. Weisser P, Krämer R, Sahm H, Sprenger GA. Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action. J Bacteriol. 1995 Jun;177(11):3351–4.

2. Biological engineering, 2021, 37(5): 1771-1793 Chinese Journal of Biotechnology, 2021, 37(5): 1771-1793.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 346
  Illegal PstI site found at 919
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 580
  Illegal PstI site found at 346
  Illegal PstI site found at 919
  Illegal NotI site found at 154
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 350
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 346
  Illegal PstI site found at 919
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 346
  Illegal PstI site found at 919
  Illegal AgeI site found at 331
  
• 1000
  COMPATIBLE WITH RFC[1000]