Engineering Success

D: In our first iteration of fuGFP-CBD fusion proteins, we placed the sequence for fuGFP upstream of each of the CBDs clos (BBa_K4488007), cex (BBa_K4488008), cipA (BBa_K4488009) and cenA (BBa_K4488010) and separated by a 2 amino acid segment. Furthermore, we added recognition sites for BsaI and BamHI at the start of our fusion protein sequences, and XhoI and BsaI sites at the end of the sequence to allow for cloning into pUS250v3 and pET28c(+) (Figure 1).

B: To build our proteins, we ordered synthetic DNA for each of our 4 fuGFP-CBDs, cloned them into pUS250v3, and transformed them into TOP10 E. coli. We expect that if the fusion proteins are successfully expressed this would be most easily discerned from the fluorescence displayed by bacteria.

T: Our initial testing included measuring whole cell fluorescence of transformed E. coli. We found that only induced cells expressing fuGFP-CBDcipA were significantly more fluorescent than uninduced cells (Figure 2). Very minor or insignificant differences in fluorescence were observed for cells expressing the other 3 fuGFP-CBD designs.

L: We hypothesised that the unexpected results were caused by misfolding of fuGFP, as we adapted the fusion proteins designs from a previous iGEM team, who used sfGFP instead of fuGFP. sfGFP folds reliably in native conditions (Pédelacq et al. 2006), whereas fuGFP has not been reported to do so. We believe that the close proximity between the CBD and fuGFP domains may interfere with protein folding of the fuGFP beta-barrel, resulting in quenching of the fluorophore and the poor fluorescence observed from most constructs.

D: Existing literature suggests flexible glycine-serine linkers can be used to improve the stability and folding of fusion proteins (Chen et al. 2013). Therefore, to improve the functionality of our fusion proteins, we redesigned the region between the two functional domains, adding a flexible (GGGGS)₃ linker between the GFP C terminus and the CBD N terminus in the second interaction of our fusion protein (Figure 3). We expected that cells expressing the new fuGFP-linker-CBD construct would be more fluorescent.

B: To build our fusion proteins, we ordered synthetic DNA for our new designs, cloned them into pUS250v3, and transformed TOP10 E. coli for testing. The new parts were named fuGFP-linker-CBDclos (BBa_K4488011), fuGFP-linker-CBDcex (BBa_K4488012), fuGFP-linker-CBDcipA (BBa_K4488013) and fuGFP-linker-CBDcenA (BBa_K4488014)

T: When testing the fluorescence of both uninduced and induced E. coli expressing the fusion proteins we found that all E. coli expressing the improved fuGFP-linker-CBD had higher average fluorescence compared to E. coli expressing the respective original fuGFP-CBDs without a linker (Figure 4). Furthermore, significant increases in fluorescence were observed between induced E. coli expressing fuGFP-linker-CBD compared to uninduced E. coli. We also found that cells expressing fuGFP-linker-CBDcipA were the most fluorescent overall.

L: Cumulatively, we learned from these results that we successfully improved our original fusion protein designs through re-engineering with the addition of a flexible linker and recorded their improved functionality through improved fluorescence (Figure 5). Further experiments show that these improved fusion proteins also bind strongly to cellulose and are eluted with glucose and we have described these findings on our main Results page.