Part:BBa_K4488020

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

Analogous to part BBa_K4488013, with a blueshifted GFP (see part BBa_K4488017).

Fusion protein of fuGFPb connected to a cipA cellulose-binding domain (CBD) via a flexible (GGGGS)3 linker. Analogous to part BBa_K4488013. This part was created using PCR with degenerate primers to mutate fuGFP-linker-CBDcipA to introduce the mutations in fuGFP creating the colour difference.

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 485 nm and peak emission occurs at 510 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 fluorescence 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 fuGFPb-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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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