Difference between revisions of "Part:BBa K3369000:Experience"
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NO detection | NO detection | ||
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As can be seen from the figure, OD540 has a good linear relationship with the 1μl to 100μl concentration, so OD540 can be used to reflect the content of NO in the sample | As can be seen from the figure, OD540 has a good linear relationship with the 1μl to 100μl concentration, so OD540 can be used to reflect the content of NO in the sample | ||
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| − | <p>Fig1. The NO change in IPTG induced cell culture. | + | <html> |
| + | <figure> | ||
| + | <img style="width:70%" src="https://parts.igem.org/File:T--Tsinghua--MLGB2.png"> | ||
| + | <figcaption> | ||
| + | <b>Figure2: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.</b> <p> The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. SpNT represents a non-targeting spacer serving as a control. </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
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| + | Fig1. The NO change in IPTG induced cell culture. | ||
| − | The change of nitrite in the bacterial solution without IPTG induction with time was measured. We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced and no IPTG induced, at the same time,we culture the bacteria with our vector Pet28-a and IPTG induced. The results are as followed. | + | Then we separately measured the change of NO over time in the NOS transformed BL21 (DE3) culture. We found that the nitrite content actually decreased over the initial period of time. The change of nitrite in the bacterial solution without IPTG induction with time was measured. We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced and no IPTG induced, at the same time,we culture the bacteria with our vector Pet28-a and IPTG induced. The results are as followed. |
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Fig3. The result of Western blot | Fig3. The result of Western blot | ||
| − | We can see that the expression of NOS-HIS gradually increased with the change of time, indicating that there was NO problem with our expression system, which also explains why the production of NO gradually increased, because in the first hours induced by IPTG, the protein expression was low, and the protein amount did not increase significantly until four hours. | + | We can see that the expression of NOS-HIS gradually increased with the change of time, indicating that there was NO problem with our expression system, which also explains why the production of NO gradually increased, because in the first hours induced by IPTG, the protein expression was low, and the protein amount did not increase significantly until four hours. |
Revision as of 13:23, 27 October 2020
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NO detection
We first prepared standard curves using standard samples of different concentrations NaNO2 and verified the linear relationship between concentration and OD540. As can be seen from the figure, OD540 has a good linear relationship with the 1μl to 100μl concentration, so OD540 can be used to reflect the content of NO in the sample
The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. SpNT represents a non-targeting spacer serving as a control.
Fig1. The NO change in IPTG induced cell culture.
Then we separately measured the change of NO over time in the NOS transformed BL21 (DE3) culture. We found that the nitrite content actually decreased over the initial period of time. The change of nitrite in the bacterial solution without IPTG induction with time was measured. We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced and no IPTG induced, at the same time,we culture the bacteria with our vector Pet28-a and IPTG induced. The results are as followed.
Fig2. The difference value of NO between IPTG induced and no IPTG induced.
As we can see, the difference value of two culture become bigger as time goes
Western blot To demonstrate that our expression system does work and to explore NOS proteins as time goes by, we performed Western blot. The culture conditions are the same as those described previously. Due to the operation, only 0-4h was detected. At the same time, GFP-His was added as the positive control.
Fig3. The result of Western blot
We can see that the expression of NOS-HIS gradually increased with the change of time, indicating that there was NO problem with our expression system, which also explains why the production of NO gradually increased, because in the first hours induced by IPTG, the protein expression was low, and the protein amount did not increase significantly until four hours.