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 has 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, fluorescent can be easily observed. We believe that this system can also be applied to various bio-detection system.

PuiChing_Macau

E.coli (sfGFP_pET11a) growth curve

       <img src="" style="width:50%">

<center><figcaption>Figure 1a. Line plot showing GFP Fluorescence emmision rate across time (hours), with different culturing mediums. Error bar representing Mean±SE.</figcaption>

     </figure></center>

       <img src="" style="width:50%">

<center><figcaption>Figure 1b. Line plot showing GFP Fluorescence emmision rate across time (hours), with different culturing mediums. Error bar representing Mean±SD. </figcaption>

     </figure></center>

To test whether E. coli can grow and express GFP in the hydroponic nutrient solutions, we compared the growth of E. coli and GFP expression in the hydroponic solution, LB medium, and water. We calculated the standard error (SE), with n=3:

       <img src="" style="width:10%">

<center><figcaption>where σ is the standard deviation, and n is the number of trials. </figcaption>

     </figure></center>

We found that the E. coli transformed with GFP is more suitable to grow in LB> hydroponic solution> pure water. As expected, the Absorbance OD600 of cell culture in LB medium is the highest. The absorbance in the hydroponic solution is lower than LB, however, it is higher than pure water, suggesting the E.coli can grow with the hydroponic solution.

The absorbance in the hydroponic solution reached a peak at the 25th hour, which shows that the E.coli grew and multiplied on the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily. These suggest E. coli can grow and survive in hydroponic solution over days.

The group's absorbance in pure water peaked at the 25th hour and decreased between the 25th to the 70th hour. This demonstrates that on the first day, the E.coli can still survive in pure water, but in the following hours the E.coli died out.

The trends of GFP fluorescence emission rate correspond to the trends of absorbance rate, showing a positive correlation between GFP protein expression and the growth of E. Coli. GFP fluorescence was highest in LB, but it is still present in hydroponic solution.

To sum up, the data indicate that E. Coli cells can survive within hydroponic systems. The data indicate that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the growth and GFP expression. (Figure 1a and 1b)

Compared to hydroponic water and LB medium, the rank of growing environments from most to least suitable E. coli transformed with GFP is LB> hydroponic water> pure water. The Absorbance rate of cell culture in LB medium is the highest, the recorded rate in the hydroponic water is lower than LB group, however, it is higher than pure water.

The Absorbance rate in LB medium is the highest and keeps increasing through out the 180 hours. This indicates that the LB medium is suitable for E.coli to grow.

The Absorbance rate in hydroponic water reached a peak at the 25th hour, which shows that the E.coli survived and multiplied in the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily.

The group in pure water has reached its peak at the 25th hour, and the rate decreases between the 25th to the 70th hour. In the following 100 hours, the rate slightly fluctuates. This demonstrates that in the first day, the E.coli can still survive in the pure water, but in the following hours the E.coli died.

To sum up, the data indicates that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the GFP expression. (Figure 1)

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]