Part:BBa_K4488019

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

Analogous to part BBa_K4488012, with a yellow shifted GFP (see part BBa_K4488016)

Fusion protein of fuGFPy 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 ~490 nm and peak emission occurs at ~510 nm (Figure 1). fuGFPy-linker-CBDcipA binds to cellulose and is eluted with glucose (figure 2). Based on SDS-PAGE, elution with glucose from cellulose yields relatively pure samples as evidenced by a single band at ~46 kDa (figure 3).

Figure 1. Absorption and emission spectra of fuGFPy-linker-cipA eluted with glucose after binding to cellulose

Figure 2. Cellulose binding test for fuGFPy-linker-CBDcipA. 250 uL of lysate containing fuGFPy-linker-CBDcipA was incubated with 250 uL of microcrystalline cellulose (0.1 g/mL) for 1 hour with mixing. The cellulose was washed with 250 uL NT buffer twice followed by the addition of 250 uL glucose (1 M) twice to elute the proteins. Bars that don’t share a letter are significantly different, bars which share a letter are not significantly different.

Figure 3. SDS-PAGE gel comparing lysate and elution samples for each colour variant of fuGFP-linker-CBDcipA. Lysate was obtained from bead beating BL21(DE3) cells expressing the proteins of interest. Elution samples were obtained by washing microcrystalline cellulose with bound proteins using glucose (1M). Distinct single bands at ~46 kDa are observed in lanes 4, 6, and 8 corresponding to fuGFP-, fuGFPy-, and fuBFP- respectively fused to CBDcipA by a linker.

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]