Difference between revisions of "Part:BBa K4205113:Experience"
| Line 21: | Line 21: | ||
<p>To verify the inducible expression of our reporter genes lacZ, we induced the bacteria at different lactate concentrations. After lysing the bacteria and performing the centrifugation, we took the supernatant and ran SDS-PAGE.</p> | <p>To verify the inducible expression of our reporter genes lacZ, we induced the bacteria at different lactate concentrations. After lysing the bacteria and performing the centrifugation, we took the supernatant and ran SDS-PAGE.</p> | ||
[[File:4-SDS.png|500px|center|thumb|left|SDS-PAGE of β-galactosidases expression in different induction conditions.]]<br> | [[File:4-SDS.png|500px|center|thumb|left|SDS-PAGE of β-galactosidases expression in different induction conditions.]]<br> | ||
| − | <p>The pre-stained protein ladder is from 10 to 180 kDa. We induced the protein expression at different lactate concentrations (1 mol/L, 10-3 mol/L, and 10-6 mol/L) and used double distilled water as the negative control. As shown in the figure | + | <p>The pre-stained protein ladder is from 10 to 180 kDa. We induced the protein expression at different lactate concentrations (1 mol/L, 10-3 mol/L, and 10-6 mol/L) and used double distilled water as the negative control. As shown in the figure above, the bands we obtained were close to the target band (113 kDa). This confirmed the expression of our target protein.</p> |
<p>Then, using ImageJ, we quantitatively analyzed the band brightness and obtained the following results: | <p>Then, using ImageJ, we quantitatively analyzed the band brightness and obtained the following results: | ||
(ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare it with the marker to get the protein expression of each lane) | (ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare it with the marker to get the protein expression of each lane) | ||
Latest revision as of 12:00, 13 October 2022
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K4205113
User Reviews
UNIQab231ca3037a5426-partinfo-00000000-QINU UNIQab231ca3037a5426-partinfo-00000001-QINU
To verify the inducible expression of our reporter genes lacZ, we induced the bacteria at different lactate concentrations. After lysing the bacteria and performing the centrifugation, we took the supernatant and ran SDS-PAGE.
The pre-stained protein ladder is from 10 to 180 kDa. We induced the protein expression at different lactate concentrations (1 mol/L, 10-3 mol/L, and 10-6 mol/L) and used double distilled water as the negative control. As shown in the figure above, the bands we obtained were close to the target band (113 kDa). This confirmed the expression of our target protein.
Then, using ImageJ, we quantitatively analyzed the band brightness and obtained the following results: (ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare it with the marker to get the protein expression of each lane)
As shown in the figure above, as the lactate concentration increased, the protein expression also increased gradually, which was consistent with our expectations.
The function of this system is to sense the elevated concentration of lactate and then detect potential mitochondrial dysfunction in autistic children. To verify this, we used filter paper, a common supply in the laboratory, to perform the experiment. First, we dropped the bacteria solution onto the filter paper. After 30 minutes, our engineering bacteria were firmly attached to the filter paper. Then we added lactate and x-gal onto the filter paper and observed the color changes.
We performed the function analysis at different lactate concentrations (1 mol/L, 10-3 mol/L, and 10-6 mol/L) and used double distilled water as the negative control.
As shown in the figure above, without the induction of lactate, the lacZ protein produced by the chassis bacteria was insufficient to color the filter paper. When adding 10-6-1 mol/L lactate, the filter paper turned blue, and as the concentration of lactate increased, the blue became darker. This confirmed that our lactate test strip was functional. However, the color change was not very significant to the naked eye, which may be related to our plasmid being a low copy, the material of filter paper, the lower activity of bacteria after fixing on the test paper, and the unsuitable reaction environment.