Difference between revisions of "Part:BBa K817002"

Latest revision as of 10:07, 20 October 2019

PfadBA

It's a fatty acyl-CoA responsive promoter which will express the downstream gene in the presence of fatty acid.

Method

To evaluate the P_fadBA promoter function, we design a P_fadBA-mRFP reporter construct. In experimental group we add oleic acid in media and compared to control group, which is the same colony and identical amount of bacteria but without addition of oleic acid. After 5 hr induction we measure the man fluorescence intensity to get the expression level of P_fadBA promoter in either group.

However, the baseline expression of the reportor gene (mRFP) downstream of P_fadBA is so high that we cannot see significant signal rise after induction of fatty acid (oleic acid). Therefore, we decide to lower the baseline expression by overexpression of FadR, an endogenous repressor of P_fadBA, whose repressive function is antagonized by fatty acyl-CoA. [3] By co-transforming constructs with P_fadBA and FadR into the bacterial platform, it is made capable of changing gene expression in response to environmental fatty acid concentration.

Protocol

1. 10 μL bacterial culture was cultured in 5 mL LB at 37。C, with suitable concentration of antibiotics shaking for 18 hr.
2. For experimental group, 480 μL of bacteria culture was transferred to 1.5 mL eppendorf tube and added with 20 μL of oleic acid and IGEPAL(detergent) in. For control group, 500 μL bacterial culture was added in 1.5 mL eppendorf tube.
3. The tubes were incubated at 37℃ 5 hr for induction.
4. 100 μL of bacterial culture was transferred tino 96-well plate. The mRFP fluorescence intensity was measured (Excitaion: 580 nm, Emmision: 610 nm).

Data

Oleic acid induction group shows higher expression of mRFP.

Conclusion

Our P_fadBA promoter can work in response to environmental fatty acid change, which acts as an important sensor for our bacterial device – secreting GLP-1 when host is fed with fatty diet.

Characterization

Team NTHU Taiwan contributed to the characterization of effects and inducing of fatty acid to E.coli and Relative Fluorescence to Anderson Promoter.
(--chiuhua 14:47, 17 October 2019 (UTC))

Team NTHU Taiwan 2019: Characterization of effects and inducing of fatty acid to E.coli and Relative Fluorescence to Anderson Promoter

Purpose

From previous study, NTU-Taida found that pFadBA can be induce by adding fatty acid. We’d like to understand more about this promoter by answering the following questions:

• Does fatty acid affect the growth of cell?
• Will higher fatty acid concentration induce more fluorescence?
• How strong is the promoter pFadBA?

Effect of Fatty acid to E. coli

We discuss the relationship between fatty acid and the growth of E. coli to check suitable conditions for future experiments. First, we check whether fatty acid affect our experiments. Second, we choose the best concentration for future experiments.
In general, relative cell concentration does not change very much. It indicates that under 0.4mM and 3.125mM fatty acid, E. coli might grow better than control. However, when the concentration is 25mM, it inhibits growth of E. coli. We can notice that low concentration of fatty acid is somehow benefit for cell growth. Therefore, we choose concentration lower than 10 mM as our highest concentration to induce the promoter in the following experiments.

Figure 1. Fluorescence detection in 96-well

Figure 2. Relative cell number with the different concentration over time

Fatty Acid Inducing to E. coli

As our previous description, pFadBA can be induced by fatty acid because of the repressor protein. We can obviously observe that with higher fatty acid level, the fluorescence level will rise. The fold change is almost 2.5 times in 0.625 mM LB, but is only 1.7 times in 0.156 mM LB. This data shows that fatty acid actually induce the promoter, and higher concentration induces more fluorescence.

Figure 3. Relative fluorescence of cell treated with different fatty acid concentration over time

Relative Fluorescence to Anderson Promoter

To know the intensity level of pFadBA expression, we compare the intensity between pFadBA and Anderson promoter. By comparing pFadBA with Anderson promoter, it demonstrates that the fluorescence level of pFadBA is much lower than the J023104 and approximately 10 percent level of J23110.

Figure 4. Comparison of pFadBA with the Anderson promoter
(J023110 with relative strength of 0.33 and J023014 with relative strength of 0.72)

Applications of BBa_K817002

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Sequence and Features