Difference between revisions of "Part:BBa K2551003:Experience"

Latest revision as of 01:00, 19 October 2021

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Please enter how you used this part and how it worked out.

EDIT by Team ULaval 2021: We ordered the coding sequence in a pET28a vector from Twist Bioscience and tested multiple conditions of protein expression in the E. coli BL21 strain.This allowed us to find the optimal condition for the expression of the part: 1mM IPTG, 37°C for 3.5h or at 0.4/1mM IPTG at 18°C for 18h (Figure 1). As expected, we observe a protein band at the expected molecular weight for this protein (95,4 kDa). However, we also see a lower weight band appears with the induction which could indicate protein degradation. The degradation is more important at 18°C and the pattern of degradation is not the same for both temperatures. To investigate this result, we looked into the literature, because this protein had already been expressed and purified prior to our experiments. In fact, this degradation was also observed by Kim et al., 2011. They also report that the catalytic activity of both fragments is still active.

As Kim et al., 2011 demonstrated, the amino residue fragment from 100 to 731(lower band at 69.4kDa) is sufficient to perform the dextranase activity of the enzyme. For a future expression and purification experiment, we would suggest to the next team to use this part to modify its sequence slightly by conserving only those residues. This truncation of the protein will provide an homogeneous protein extract and allow the team to use the dextranase without worrying about its different forms.

Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.

Applications of BBa_K2551003

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UNIQ64a9aed40edb12ea-partinfo-00000000-QINU UNIQ64a9aed40edb12ea-partinfo-00000001-QINU

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 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+EDIT by Team ULaval 2021: We ordered the coding sequence in a pET28a vector from Twist Bioscience and tested multiple conditions of protein expression in the E. coli BL21 strain.This allowed us to find the optimal condition for the expression of the part: 1mM IPTG, 37°C for 3.5h or at 0.4/1mM IPTG at 18°C for 18h (Figure 1). As expected, we observe a protein band at the expected molecular weight for this protein (95,4 kDa). However, we also see a lower weight band appears with the induction which could indicate protein degradation. The degradation is more important at 18°C and the pattern of degradation is not the same for both temperatures. To investigate this result, we looked into the literature, because this protein had already been expressed and purified prior to our experiments. In fact, this degradation was also observed by Kim et al., 2011. They also report that the catalytic activity of both fragments is still active.
+As Kim et al., 2011 demonstrated, the amino residue fragment from 100 to 731(lower band at 69.4kDa) is sufficient to perform the dextranase activity of the enzyme. For a future expression and purification experiment, we would suggest to the next team to use this part to modify its sequence slightly by conserving only those residues. This truncation of the protein will provide an homogeneous protein extract and allow the team to use the dextranase without worrying about its different forms.
+Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.
 ===Applications of BBa_K2551003===