Designed by: Donovan Wu, Jackie Yau, Oliver Nicholls, Jasmin Li Group: iGEM22_Sydney_Australia (2022-09-30)
Fusion of free-use GFP with CBDcipA (cellulose-binding domain) at the C-terminal end with a linker
The construct can be cloned into an expression vector such as pET28c in E.coli to produce a fusion protein of fuGFP with CBDcipA. The fuGFP sequence is towards the N terminus of the protein with CBDcipA (BBa_K4488024 ) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon.
Usage and Biology
Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-linker-CBDcipA.
Figure 1: Fluorescence measurements of filter paper binding test with fuGFP-linker-CBDs. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measurements show that lysate with fusion proteins with the added linker exhibit more fluorescence than control cell lysate and makes the filter disks fluorescent.. fuGFP-linker-CBDcipA causes the greatest gain in fluorescence by paper filter disks.
The linker improved the solubility and fluorescence of the sequence. Below is the fluorescence assay depicting a higher reading compared to our previous construct BBa_K4488009 :
Figure 2: Fluorescence readings of fuGFP-linker-CBDs compared to fuGFP-CBDs in different E.coli plasmids. The uninduced and induced plasmids are also compared.
Binding to Cellulose
Furthermore, expression using the BL21(DE3)-pET28c(+) system results in high yields of protein which can be purified by binding to cellulose and eluting using glucose. Expression and cell fluorescence in BL21(DE3) is shown in figure 3. Using a cellulose binding test we showed that it causes cellulose to gain fluorescence and is eluted using glucose (figures 4 and 7). We also tested other eluents, such as glycerol and maltose, at varying concentrations and concluded the optimum condition to reach maximum fluorescence was using 1M glucose (figure 5 and 6). Finally, SDS-PAGE shows that purified samples of fuGFP-linker-CBDcipA is obtained from elution after binding cellulose (figure 8).
Figure 3: LB-kanamycin patch plates of BL21(DE3)-pET28c(+)-fuGFP-linker-CBDs. Kanamycin was added to 50 μg/mL concentration and 10 μL 0.5 M IPTG was added. Plates were incubated at 37 C for 20 h.
Figure 4: Fluorescence measurements of fuGFP-linker-CBDcipA bound to microcrystalline cellulose. 250 μL of cell lysate containing fuGFP-linker-CBDcipA was incubated with 250 μL of microcrystalline solution (10% w/v) for 1 h with rotation. The supernatant was removed and the cellulose was washed twice with 250 μL of NT buffer and also removed by centrifugation each time. Bars represent means ± SE, n = 3. Results of a Tweedie family GLM showed a significant interaction between construct and treatment (χ23 = 709.7, p < 0.001). Bars that don’t share a letter are significantly different based on Tukey-Kramer post-hoc contrast. Microcrystalline cellulose gains significant fluorescence that is not washed off when fuGFP-linker-CBDcipA is added in comparison to a fuGFP control.
Figure 5: Fluorescence measurements of elutions of fuGFP-linker-CBDcipA from microcrystalline cellulose under different conditions. Cell lysate containing fuGFP-linker-CBDcipA was incubated with 250 uL of microcrystalline solution (10% w/v) for 1 h with rotation. The supernatant was removed and the cellulose was washed twice with 250 uL NT buffer. Fusion proteins were then eluted from the cellulose by washing three times with 250 uL of different conditions. Results show fluorescence of elution fractions increases with each repetition and using 1 M glucose results in the greatest fluorescence in the third elution.
Figure 6: Fluorescence measurements of elutions of fuGFP-linker-CBDcipA from microcrystalline cellulose comparing the different concentrations of the eluents. Cell lysate containing fuGFP-linker-CBDcipA was incubated with 250 uL of microcrystalline solution (10% w/v) for 1 h with rotation. The supernatant was removed and the cellulose was washed twice with 250 uL NT buffer. Fusion proteins were then eluted from the cellulose by washing three times with 250 uL of different conditions. 1M concentrations demonstrated the greatest fluorescence.
Figure 7: luorescence measurements of fuGFP-linker-CBDcipA bound to microcrystalline cellulose. 250 μL of cell lysate containing fuGFP-linker-CBDcipA was incubated with 250 μL of microcrystalline solution (10% w/v) for 1 h with rotation. The supernatant was removed and the cellulose was washed twice with 250 μL of NT buffer and also removed by centrifugation each time. Bars represent means ± SE, n = 3. Results of a Tweedie family GLM showed a significant interaction between construct and treatment (χ23 = 709.7, p < 0.001). Bars that don’t share a letter are significantly different based on Tukey-Kramer post-hoc contrast. Microcrystalline cellulose gains significant fluorescence that is not washed off when fuGFP-linker-CBDcipA is added in comparison to a fuGFP control.
Figure 8: Fluorescence measurements of elutions of fuGFP-linker-CBDcipA from microcrystalline cellulose using 1 M glucose. Cell lysate containing fuGFP-linker-CBDcipA was incubated with 250 μL of microcrystalline solution (10% w/v) for 1 h with rotation. The supernatant was removed and the cellulose was washed twice with 250 μL NT buffer. Fusion proteins were then eluted from the cellulose by washing four times with 250 μL of glucose (1 M). Bars represent means ± SE, n = 3. Results of a Tweedie family GLM showed a significant interaction between elution step and treatment (χ23 = 3559.3, p < 0.001). Bars that don’t share a letter are significantly different based on Tukey-Kramer post-hoc contrast. Results show fluorescence of elution fractions increases with each repetition and the final fluorescence of microcrystalline cellulose with bound fusion proteins is halved at the end in comparison to cellulose washed with only NT buffer an additional four times as a control.
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