Difference between revisions of "Part:BBa K2938004"

Revision as of 22:03, 7 October 2022

P20

Description

p20 is a 20 kDa protein which can be isolated from Bacillus thuringiensis subsp. israelensis (Bti). It is a chaperone, which increases the production of Cry11Aa while expressed, and leads to higher mosquito larvicidal activity [2].

Amplification

PCR amplification of P20 by PCR.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 264
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 264
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 264
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 264
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 264
1000
COMPATIBLE WITH RFC[1000]

Contribution by iGEM2022 Guelph - The effect of P20 on cell viability and stability of Cyt1Aa

Bacillus thuringiensis subsp. Israelensis encodes crystal (Cry) and cytotoxic (Cyt) proteins which lyse target insect cells through a similar process. The expression of these Cry and Cyt proteins can be dependent on the coexpression of accessory helper proteins (Shao et al., 2001). In this case, the helper protein P20 increases the expression of Cry proteins (Cry1Ac, Cry4Aa, Cry4Ba, and Cry11Aa) and is necessary to observe colony formation in its original host and E.coli (Shao et al., 2001).

The P20 helper protein is a 20 kDa protein that was first identified in 1987 by Mclean and Whiteley and following this discovery, it was hypothesized that this protein allows for the crystalization of Cyt1Aa, Cry4Ba, Cry11Aa, and Cry4Aa. Manasherob et al. (2001), later reported that plasmid constructs expressing only Cyt1Aa showed reduced colony-forming ability and Cyt1Aa protein expression (Manasherob et al., 2001). While Cyt1Aa and P20 expressing plasmid constructs showed higher Cyt1Aa protein expression and cell viability in recombinant E.coli cells (Manasherob et al., 2001). This confirms that P20 stabilizes Cyt1Aa and inhibits cell death. Although the exact mechanism by which P20 does this was not known in 1987, Manasherob et al. (2001) proposed that pre-mature activation of Cyt1Aa and lethality was a result of incomplete degradation of this Cyt protein. P20 binds to and prevents the degradation of Cyt1Aa, which ultimately results in the viability of the cell (E.coli and Bacillus thuringiensis cells) (Manasherob et al., 2001). This is an important step in the expression of Cyt1Aa because this Cyt protein does not form inclusion bodies that further protect the cell from Cyt1Aa’s cytotoxic effects (Manasherob et al., 2001). For these reasons, the coexpression of P20 must be considered when expression of Cyt1Aa is desired as this protein stabilizes the crystal formation and protects cells from the lethal effects of this protein.

References

Manasherob, Robert, et al. “Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of cyt1aa from Bacillus Thuringiensis Subsp. Israelensis in Escherichia Coli.” Current Microbiology, vol. 43, no. 5, 3 Apr. 2001, pp. 355–364., https://doi.org/10.1007/s002840010316.

Shao, Z., Liu, Z., & Yu, Z. (2001). Effects of the 20-kilodalton helper protein on cry1ac production and spore formation in bacillus thuringiensis. Applied and Environmental Microbiology, 67(12), 5362–5369. https://doi.org/10.1128/aem.67.12.5362-5369.2001.

@@ Line 13: / Line 13: @@
 <partinfo>BBa_K2938004 SequenceAndFeatures</partinfo>
+===Contribution by iGEM2022 Guelph - The effect of P20 on cell viability and stability of Cyt1Aa===
+Bacillus thuringiensis subsp. Israelensis encodes crystal (Cry) and cytotoxic (Cyt) proteins which lyse target insect cells through a similar process. The expression of these Cry and Cyt proteins can be dependent on the coexpression of accessory helper proteins (Shao et al., 2001). In this case, the helper protein P20 increases the expression of Cry proteins (Cry1Ac, Cry4Aa, Cry4Ba, and Cry11Aa) and is necessary to observe colony formation in its original host and E.coli (Shao et al., 2001).
+The P20 helper protein is a 20 kDa protein that was first identified in 1987 by Mclean and Whiteley and following this discovery, it was hypothesized that this protein allows for the crystalization of Cyt1Aa, Cry4Ba, Cry11Aa, and Cry4Aa. Manasherob et al. (2001), later reported that plasmid constructs expressing only Cyt1Aa showed reduced colony-forming ability and Cyt1Aa protein expression (Manasherob et al., 2001). While Cyt1Aa and P20 expressing plasmid constructs showed higher Cyt1Aa protein expression and cell viability in recombinant E.coli cells (Manasherob et al., 2001). This confirms that P20 stabilizes Cyt1Aa and inhibits cell death. Although the exact mechanism by which P20 does this was not known in 1987, Manasherob et al. (2001) proposed that pre-mature activation of Cyt1Aa and lethality was a result of incomplete degradation of this Cyt protein. P20 binds to and prevents the degradation of Cyt1Aa, which ultimately results in the viability of the cell (E.coli and Bacillus thuringiensis cells) (Manasherob et al., 2001). This is an important step in the expression of Cyt1Aa because this Cyt protein does not form inclusion bodies that further protect the cell from Cyt1Aa’s cytotoxic effects (Manasherob et al., 2001). For these reasons, the coexpression of P20 must be considered when expression of Cyt1Aa is desired as this protein stabilizes the crystal formation and protects cells from the lethal effects of this protein.
+==References==
+Manasherob, Robert, et al. “Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of cyt1aa from Bacillus Thuringiensis Subsp. Israelensis in Escherichia Coli.” Current Microbiology, vol. 43, no. 5, 3 Apr. 2001, pp. 355–364., https://doi.org/10.1007/s002840010316.
+Shao, Z., Liu, Z., & Yu, Z. (2001). Effects of the 20-kilodalton helper protein on cry1ac production and spore formation in bacillus thuringiensis. Applied and Environmental Microbiology, 67(12), 5362–5369. https://doi.org/10.1128/aem.67.12.5362-5369.2001.
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