Difference between revisions of "Part:BBa K2817004"
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We inserted this part into plasmid pet-28a. So the expression of this part can be induced by IPTG. We transformed the plasmid of myrosinase into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added and induced at 16 ℃ for 16 h. The bacterial cell lysis was then performed to detect the expression of myrosinase by SDS-PAGE (Figure 1). The sequence of the myrosinase enzyme was optimized based on the K12 strain and the position of the strip is almost correct, so we believe we have successfully expressed it. The myrosinase can convert glucosinolates contained in cruciferous vegetables into sulforaphane which has well-known anti-cancer activity. A literature earlier this year (https://doi.org/10.1038/s41551-017-0181-y) has validated the activity of myrosinase expressed by E. coli Nissle 1917 in vivo and in vitro. So we believe that the myrosinase we expressed can work. For more details about this transferase related information, please see our project design. | We inserted this part into plasmid pet-28a. So the expression of this part can be induced by IPTG. We transformed the plasmid of myrosinase into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added and induced at 16 ℃ for 16 h. The bacterial cell lysis was then performed to detect the expression of myrosinase by SDS-PAGE (Figure 1). The sequence of the myrosinase enzyme was optimized based on the K12 strain and the position of the strip is almost correct, so we believe we have successfully expressed it. The myrosinase can convert glucosinolates contained in cruciferous vegetables into sulforaphane which has well-known anti-cancer activity. A literature earlier this year (https://doi.org/10.1038/s41551-017-0181-y) has validated the activity of myrosinase expressed by E. coli Nissle 1917 in vivo and in vitro. So we believe that the myrosinase we expressed can work. For more details about this transferase related information, please see our project design. | ||
| − | https://static.igem.org/mediawiki/2018/a/ae/T--NEU_China_A-- | + | https://static.igem.org/mediawiki/2018/a/ae/T--NEU_China_A--results-8.png |
Figure 1. SDS-PAGE analyses on bacterial lysate to detect myrosinase. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃. | Figure 1. SDS-PAGE analyses on bacterial lysate to detect myrosinase. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃. | ||
Revision as of 15:57, 16 October 2018
Myrosinase (horseradish)
Myrosinase is present in cruciferous plants such as broccoli, Brussels sprouts and Celery cabbage. It can convert the precursor glucosinolates in cruciferous plants into sulforaphane, a well-known anticancer substance. Sulforaphane is an activator of Nrf2, which induces Nrf2 activation and translocation into the nucleus, binding to the antioxidant response element (ARE) to promote transcriptional activation of the phase II metabolic enzyme gene. As a result, sulforaphane, on the one hand, can eliminate cancer cells by inducing phase II reaction; on the other hand, it can alleviate inflammation by inducing antioxidant enzymes (such as HO-1 and SOD) to resist oxidative stress. The horseradish enzyme derived from horseradish has good catalytic activity at body temperature and in vivo pH.
Usage and Biology
We inserted this part into plasmid pet-28a. So the expression of this part can be induced by IPTG. We transformed the plasmid of myrosinase into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added and induced at 16 ℃ for 16 h. The bacterial cell lysis was then performed to detect the expression of myrosinase by SDS-PAGE (Figure 1). The sequence of the myrosinase enzyme was optimized based on the K12 strain and the position of the strip is almost correct, so we believe we have successfully expressed it. The myrosinase can convert glucosinolates contained in cruciferous vegetables into sulforaphane which has well-known anti-cancer activity. A literature earlier this year (https://doi.org/10.1038/s41551-017-0181-y) has validated the activity of myrosinase expressed by E. coli Nissle 1917 in vivo and in vitro. So we believe that the myrosinase we expressed can work. For more details about this transferase related information, please see our project design.
Figure 1. SDS-PAGE analyses on bacterial lysate to detect myrosinase. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃.
[1] Ho CL, Tian HQ.et al. 2018 Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature Biomedical Engineering. 2, pages27–37
[2] Tortorella, S. M., Royce, S. G., Licciardi, P. V. & Karagiannis, T. C. Dietary sulforaphane in cancer chemoprevention: the role of epigenetic regulation and HDAC inhibition. Antioxid. Redox Signal. 22, 1382–1424 (2015).
[3] Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW; Talalay P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keapl sensor modified by inducers. Proc Natl Acad Sci U S A. 2004;101(7):2040一2045.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 139
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 368
Illegal BsaI site found at 1397