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Using T7 strong promoter to express OprF, derived from Pseudomonus areuginosa, would strongly improve the bacterial permeability.
Using T7 strong promoter to express OprF, derived from Pseudomonus areuginosa, would strongly improve the bacterial permeability.
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== T7 ==
== T7 ==
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Information of T7:see <html><a href="https://parts.igem.org/Part:BBa_I712074" target="_blank">BBa_I712074</a>
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Information of T7:see [[Part:BBa_I712074 | BBa_I712074]]
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== OprF ==
== OprF ==
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Informtion of OprF:see [[Part:BBa_K1593209 | BBa_K1593209]]
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Informtion of OprF:see<html><a href="https://parts.igem.org/Part:BBa_K1593209" target="_blank">BBa_K1593209</a>
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== Growth Characterization ==
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This is in order to demonstrate that the overexpression of porin won't severely affect the growth of bacteria, at least it won't significantly inhibit bacteria growth and become as kill switch.
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[[File:4900.jpg]]
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== Growth Characterization ==
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== ONPG Assay ==
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We firstly characterized the growth rate of genetically modified CACCI along with wild type BL21 using optical density at 600 nm, which is called OD 600, in order to demonstrate that the overexpression of porin(OprF with T7,[BBa_K1593209](http://tower.im)) or viroporin(SCVE with T7, ([BBa_K1593667](https://parts.igem.org/Part:BBa_K1593667)) won't severely affect the growth of bacteria, at least it won't significantly inhibit bacteria growth and become as kill switch.
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This is to show the absorption capability of the bacteria is improved.
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All bacteria are culture previously in LB medium about 12 h. And then measurement of bacteria growth through time began at 8 A.M. the other day.
As the images illustrated above, we found *E. coli* BL21 wild type has the best growth characteristics with a maximal OD600=3.427. Whereas *E. coli* BL21 with OprF ([BBa_K1593210](https://parts.igem.org/Part:BBa_K1593210)) has a relatively low optical density as its OD600 after 12 h is 2.808 compared to the wildtype. In the same way, characterization of *E. coli* BL21 with SCVE, https://parts.igem.org/Part:BBa_K1593667 showed a slightly small OD600, as its finally turn to 2.835. *E. coli* BL21 with T7-OprF ([BBa_K1593210](https://parts.igem.org/Part:BBa_K1593210)) and *E. coli* BL21 with T7-SCVE, ([BBa_K1593667](https://parts.igem.org/Part:BBa_K1593667)) are showing 19% decreased maximal cell density and 18.5% decreased maximal cell density respectively in comparison to wild type.
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[[File:Ustc-onpg88.png]]
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Consequently, the result implies that genetic modification on bacterial permeability, at least overexpression of OprF and SCVE with strong promoter T7, won't significantly influence bacterial growth. Thus, genetic modified bacteria are able to be used in the following experiments.
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Later on, we will characterize the basic permeability capability of *E. coli* BL21 with T7-OprF ([BBa_K1593210](https://parts.igem.org/Part:BBa_K1593210)) and *E. coli* BL21 with T7-SCVE, ([BBa_K1593667](https://parts.igem.org/Part:BBa_K1593667)).
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===Usage and Biology===
===Usage and Biology===
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Latest revision as of 14:56, 18 September 2015
T7-OprF
Using T7 strong promoter to express OprF, derived from Pseudomonus areuginosa, would strongly improve the bacterial permeability.
This is in order to demonstrate that the overexpression of porin won't severely affect the growth of bacteria, at least it won't significantly inhibit bacteria growth and become as kill switch.
ONPG Assay
This is to show the absorption capability of the bacteria is improved.
