Difference between revisions of "Part:BBa K2762014"

Latest revision as of 00:13, 12 October 2022

P_asr-B0034-sfGFP

Background

The asr promoter was first described by Suziedeliene et al. in 1999. They showed that asr is induced under low pH which is about pH 4.8, and it is controlled by the phoBR system. From the article they have published, the promoter is named as acid shock RNA (asr) promoter due to the RNA that has been transcribed after putting the E. coli into a low pH condition.

In 2007 Ogasawara et al2. found out that there is another regulatory system that controlling asr transcription by using SELEX to find the binding sequences of PhoQP-RstBA. Hence the asr promoter is directly controlled by two different systems, the phoBR system activated through low inorganic phosphate and the RstAB system sensing the pH while it is controlled by PhoQP-system activated by low Mg²⁺ concentrations.

Function of asr promoter in sfGFP reporting system

To expand the possible application of asr promoter, iGEM NCKU_Tainan 2018 put it together with sfGFP and tested this reporting system with more delicate pH gradings since a green fluorescent would be expressed once the promoter has been activated. In conclusion, we could monitor the pH in the surrounding medium in our device at any time by observing the color change of the medium.

Characterization

Colony PCR of finished construct

After finishing the P_asr biobrick construction, colony PCR is run to check the success of ligation. The length of the DNA is verified with agarose gel electrophoresis.

Charaterization of promoter with fluorescent intensity measurement

P_asr is reported to be induced in acidic condition. We think that it can be used to report the abnormal acidity of the medium. We thus determine to measure the fluorescent intensity in a short period of time. We first incubated the bacteria to log phase (within 2 hour) with LB medium. We then centrifuged the broth and suspended the pellet with pH modified M9 medium (the pH value is modified with 1M HCl). We then took the sample and incubate in the 96 well and measure its fluorescent intensity for every 3 minutes. We found out that the promoter P_asr will be induced at the pH value below four within 30 minutes. The different fluorescent intensity can be observed within 30 minutes. The fluorescent had the peak at pH value of 4.25.

Fig 2. The data shows the fluorescent intensity (absorbance: 485 nm, excitation: 535 nm) expressed by P_asr in different pH.

Based on the data shown above, we could conclude that P_asr is an acidic promoter as it has a high expression of fluorescent at pH 4.25 and pH 5. The results show that P_asr constructed pH sensing system can be used as an alert. When the medium turns acidic, fluorescence can be easily observed. We believe that this system can also be applied to various bio-detection systems.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 314
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2762014 short</partinfo>
-===Characterization===
+===Background===
-The strain that we used is carrying asr promoter, a pH-responsive promoter which is native to <i>E. coli</i> and it could induce transcription in acidic conditions. Besides, we clone a sfGFP gene downstream of this promoter which could express green fluorescent once the promoter has been activated.
+The <i>asr</i> promoter was first described by Suziedeliene et al. in 1999. They showed that <i>asr</i> is induced under low pH which is about pH 4.8, and it is controlled by the <i>phoBR</i> system. From the article they have published, the promoter is named as acid shock RNA (asr) promoter due to the RNA that has been transcribed after putting the <i>E. coli</i> into a low pH condition.
-In conclusion, when the color of the medium turns from turbid yellow to green,  it indicates the pH of the medium is too low so the medium should be changed as it is not suitable for our <i>E. coli</i> to grow.
+In 2007 Ogasawara et al2. found out that there is another regulatory system that controlling <i>asr</i> transcription by using SELEX to find the binding sequences of <i>PhoQP-RstBA</i>. Hence the <i>asr</i> promoter is directly controlled by two different systems, the <i>phoBR</i> system activated through low inorganic phosphate and the <i>RstAB</i> system sensing the pH while it is controlled by <i>PhoQP</i>-system activated by low Mg<sup>2+</sup> concentrations.
+===Function of asr promoter in sfGFP reporting system===
+To expand the possible application of asr promoter, iGEM NCKU_Tainan 2018 put it together with <i>sfGFP</i> and tested this reporting system with more delicate pH gradings since a green fluorescent would be expressed once the promoter has been activated. In conclusion, we could monitor the pH in the surrounding medium in our device at any time by observing the color change of the medium.
+==Characterization==
+===Colony PCR of finished construct===
+After finishing the P<sub>asr</sub> biobrick construction, colony PCR is run to check the success of ligation. The length of the DNA is verified with agarose gel electrophoresis.
+[[File:T--NCKU Tainan--part BBa K2762014.png|200px|centre]]
+===Charaterization of promoter with fluorescent intensity measurement===
+P<sub>asr</sub> is reported to be induced in acidic condition. We think that it can be used to report the abnormal acidity of the medium. We thus determine to measure the fluorescent intensity in a short period of time. We first incubated the bacteria to log phase (within 2 hour) with LB medium. We then centrifuged the broth and suspended the pellet with pH modified M9 medium (the pH value is modified with 1M HCl). We then took the sample and incubate in the 96 well and measure its fluorescent intensity for every 3 minutes. We found out that the promoter P<sub>asr</sub> will be induced at the pH value below four within 30 minutes. The different fluorescent intensity can be observed within 30 minutes. The fluorescent had the peak at pH value of 4.25.
+[[File:T--NCKU_Tainan--Results fig 21.png|460px|centre]]
+Fig 2. The data shows the fluorescent intensity (absorbance: 485 nm, excitation: 535 nm) expressed by P<sub>asr</sub> in different pH.
+Based on the data shown above, we could conclude that P<sub>asr</sub> is an acidic promoter as it has a high expression of fluorescent at pH 4.25 and pH 5. The results show that P<sub>asr</sub> constructed pH sensing system can be used as an alert. When the medium turns acidic, fluorescence can be easily observed. We believe that this system can also be applied to various bio-detection systems.
-The asr promoter was first described by Suziedeliene et al. in 1999. They showed that asr is induced under low pH which is about pH 4.8, and it is controlled by the phoBR system. From the article they have published, the promoter is named as acid shock RNA (asr) promoter due to the RNA that has been transcribed after putting the E. coli into a low pH condition.
-In 2007 Ogasawara et al2. found out that there is another regulatory system that controlling asr transcription by using SELEX to find the binding sequences of PhoQP-RstBA. Hence the asr promoter is directly controlled by two different systems, the PhoBR system activated through low inorganic phosphate and the RstAB system sensing the pH while it is controlled by PhoQP-system activated by low Mg<sup>2+</sup> concentrations.
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