Difference between revisions of "Part:BBa K4156128"
| (One intermediate revision by the same user not shown) | |||
| Line 5: | Line 5: | ||
HlyE is a toxin, by breaking the cell membrane of the tumor, the tumor cells are destroyed. In our experiments, we used HlyE as a tumor treatment factor and it is connected with plac promoter to observe of bacterial adhesion effect. | HlyE is a toxin, by breaking the cell membrane of the tumor, the tumor cells are destroyed. In our experiments, we used HlyE as a tumor treatment factor and it is connected with plac promoter to observe of bacterial adhesion effect. | ||
| − | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here --> |
| + | |||
| + | ===Characterization=== | ||
==Western blot== | ==Western blot== | ||
To verify the extracellular secretion of HlyE, we constructed an AE strain by fusing his tag at the C-terminus of HlyE. Then, the AE strain (HlyE with his tag) was inoculated in 50 ml of LB medium containing the corresponding antibiotics and cultured overnight at 37 °C. Then, 5 ml of the culture was centrifuged and the supernatant was collected. The supernatant was concentrated using the TCA precipitation method (25% TCA, -20°C, 1h) to isolate the total protein. Finally, the expression of HlyE was detected by western blot. The results showed that the constitutive promoter could secrete HlyE under both inducible and non-inducible conditions, while the lactate (plldR), pH (pCadc) and hypoxia (pPepT) inducible reporters could only secrete HlyE under inducible conditions and not under non-inducible conditions. indicated that our constructed AE strain could well cope with environmental induction and secrete HlyE in the tumor microenvironment It was shown that our AE strain could respond well to environmental induction and secrete HlyE in the tumor microenvironment, thus killing cancer cells without harming other normal cells. | To verify the extracellular secretion of HlyE, we constructed an AE strain by fusing his tag at the C-terminus of HlyE. Then, the AE strain (HlyE with his tag) was inoculated in 50 ml of LB medium containing the corresponding antibiotics and cultured overnight at 37 °C. Then, 5 ml of the culture was centrifuged and the supernatant was collected. The supernatant was concentrated using the TCA precipitation method (25% TCA, -20°C, 1h) to isolate the total protein. Finally, the expression of HlyE was detected by western blot. The results showed that the constitutive promoter could secrete HlyE under both inducible and non-inducible conditions, while the lactate (plldR), pH (pCadc) and hypoxia (pPepT) inducible reporters could only secrete HlyE under inducible conditions and not under non-inducible conditions. indicated that our constructed AE strain could well cope with environmental induction and secrete HlyE in the tumor microenvironment It was shown that our AE strain could respond well to environmental induction and secrete HlyE in the tumor microenvironment, thus killing cancer cells without harming other normal cells. | ||
| Line 16: | Line 18: | ||
</html> | </html> | ||
| − | + | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4156128 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4156128 SequenceAndFeatures</partinfo> | ||
Latest revision as of 03:34, 11 October 2022
plac-HlyE
HlyE is a toxin, by breaking the cell membrane of the tumor, the tumor cells are destroyed. In our experiments, we used HlyE as a tumor treatment factor and it is connected with plac promoter to observe of bacterial adhesion effect.
Characterization
Western blot
To verify the extracellular secretion of HlyE, we constructed an AE strain by fusing his tag at the C-terminus of HlyE. Then, the AE strain (HlyE with his tag) was inoculated in 50 ml of LB medium containing the corresponding antibiotics and cultured overnight at 37 °C. Then, 5 ml of the culture was centrifuged and the supernatant was collected. The supernatant was concentrated using the TCA precipitation method (25% TCA, -20°C, 1h) to isolate the total protein. Finally, the expression of HlyE was detected by western blot. The results showed that the constitutive promoter could secrete HlyE under both inducible and non-inducible conditions, while the lactate (plldR), pH (pCadc) and hypoxia (pPepT) inducible reporters could only secrete HlyE under inducible conditions and not under non-inducible conditions. indicated that our constructed AE strain could well cope with environmental induction and secrete HlyE in the tumor microenvironment It was shown that our AE strain could respond well to environmental induction and secrete HlyE in the tumor microenvironment, thus killing cancer cells without harming other normal cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 359
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]