Part:BBa_K4488026

Fusion of free-use GFP-alanine with CBDcipA (cellulose-binding domain) at the C-terminal end with a

Analogous to part BBa_K4488025, with a blue shifted free use GFP that absorbs blue light rather than UV (see part BBa_K4488018).

Usage and Biology

This part contains restriction sites making it suitable for traditional cloning with BamHI and XhoI as well as golden gate cloning with BsaI. The 2022 USYD Sydney Australia team was successful in expressing this protein with the BL21(DE)-pET28c(+) system. This fusion protein will fluoresce under UV and will bind to cellulose. Based on plate reader measurements, peak excitation occurs at 480 nm and peak emission occurs at 505 nm (Figure 1). A comparison of its spectra with other fuGFPvar-linker-CBDcipA is shown in figure 2. We also found that when treating cellulose with cell lysate containing this fusion protein, there is a significant gain in fluorescence which suggests the protein has functional affinity for cellulose (figure 3). However, based on measurements we were unable to confirm whether a significant yield of the protein was obtained when attempting to elute the fusion protein from cellulose.

Figure 1. Absorption and emission spectra of fuGFPb-linker-cipA measured from cell lysate

Figure 2.Excitation and emission spectra measured from fuGFP-, fuGFPb-, fuGFPy-, fuGFPa-, fuBFP-, linked to CBDcipA. Samples of the fuGFP-, fuGFPy-, fuBFP-fusion proteins were eluted with glucose after binding to cellulose and their spectra were measured in a plate reader using our plate reader protocol. Samples of fuGFPb- and fuGFPa- fusion proteins were measured from cell lysates

Figure 3. Cellulose binding test for fuGFPa-linker-cipA. 250 uL of cell lysate containing the fusion protein was incubated with 250 uL microcrystalline cellulose (0.1g/mL) for 1 hour with shaking. The cellulose was washed with 250 uL NT buffer twice followed by 4 additions of glucose (1 M) for elution

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1361
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 151
  
• 1000
  COMPATIBLE WITH RFC[1000]