Part Improvement

Background
This part is the coding region for an endoglucanse from Ruminiclostridium thermocellum that was previously created by the iGEM16_Dalhousie_NS_Halifax (BBa_K2160000). Its function is to cleave internal beta-1,4-D-glycosidic bonds in crystalline cellulose to release the disaccharide cellobiose.

Improvement
We improved the endoglucanase part by adding a C-terminal His-tag and an N-terminal PelB sequence. The C-terminal His-tag allows identification via western blot or immuno-fluorescence, and protein purification. The PelB sequence is a localization sequence that traffics the protein to the periplasm (Sockolosky & Szoka, 2013). This is especially important for our project because we need to get all the enzymes out of the E. coli to digest cellulose.
Using a western blot to probe for the His-Tag, we were able to show expression of our optimized endoglucanase. The main species traveled at 46 kDa, which was the predicted migration of endoglucanase with a HIS-tag and PelB sequence. A secondary, smaller species was seen at ~30 kDa. The 30 kDa species can be explained due to a second methionine codon with an imperfect ribosomal binding sequence 5-10 bp upstream from the Met codon. Figure 1. Western blot probing for 6xHis Tag

Part Number
Our part can be found here: BBa_K2160000

References

Sockolosky, J. and Szoka, F. (2013). Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expression and Purification, 87(2), pp.129-135.