Part:BBa_K817033
PfadBA-RBS-mRFP
It's the PfadBA reporter.
Method
To evaluate the PfadBA promoter function, we design a PfadBA-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 PfadBA promoter in either group.
However, the baseline expression of the reportor gene (mRFP) downstream of PfadBA 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 PfadBA, whose repressive function is antagonized by fatty acyl-CoA. [3] By co-transforming constructs with PfadBA and FadR into the bacterial platform, it is made capable of changing gene expression in response to environmental fatty acid concentration.
Protocol
- 10 μL bacterial culture was cultured in 5 mL LB at 37。C, with suitable concentration of antibiotics shaking for 18 hr.
- 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.
- The tubes were incubated at 37℃ 5 hr for induction.
- 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 PfadBA 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.
Improvement
Designed by NTHU_Taiwan 2019. (See the part at BBa_K3040007)
Background
This has an improvement on the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817033). However, according to their result, this promoter has a massive leakage, which has very high value of downstream reporter gene baseline expression. Besides, the fold change after 0.04% of oleic acid induction has only achieved 1.67-fold, considered no significant signal rise. Thus, we coupled an endogenous thioesterase coding gene with this promoter. By doing so, we are able to perform a tunable and dynamic gene expression.
Mechanism
Tes A is a heterologous thioesterase, which is able to hydrolyze fatty acyl-CoAs to free fatty acids. Here, we combine Tes A with pFadBA (wild type) together as a new promoter, Tes A pFadBA, to see if the sensitivity range of fatty acid concentration lowers due to the increase of endogenous fatty acid.
In the microbial, carbon source such as glucose or fatty acid will be metabolized to acetyl-CoA. When fatty acid is needed to be catabolized into other macromolecules, the acetyl-CoA will be converted into Acyl-ACP and finally formed free fatty acid. The following is the detailed mechanism of the biosynthetic and degradation pathway of fatty acid. [1]
Nevertheless, the accumulation of Acyl-ACP will negatively inhibit the conversion of malonyl-CoA to Acyl-ACP and thus repress biosynthesis of free fatty acid. Thus, endogenous TesA gene which encodes a thioesterase will hydrolyze these Acyl-ACP and subsequently produce a significant level of free fatty acid.
Concept of design
As you know, high concentration of fatty acid will promote β-oxidation but not synthesis of fatty acid. However, overexpression of TesA can help the E. coli to deplete the Acyl-ACP and thus rescue the production of free fatty acid. The more fatty acid present, the more acyl-CoA can be converted and thus the higher transcription rate can pfadBA can achieved. The following was the mechanism proposed.
Some previous literature has reported that the strain carries TesA overexpression is capable of showing 10 to 25-fold of fluorescence than the native promoter [2]. This result matched our proposed model.
Result
As we predicted that the fluorescence fold change after induction of fatty acid will be greater as tesA has produce more fatty acid. The result shows that the fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden.
Future work
According to the previous research, the fold change should be able to reached 10 to 25-fold. We deduce that the problem is we used a weaker promoter placed before TesA, thus the fatty acid produced endogenously is fewer than previously reported. Hence, we proposed to insert the TesA sequence directly at the downstream of pfadBA promoter and made it regulated by this promoter. Once we have added the fatty acid, then pfadBA promoter will be activated, RFP and tesA will be produced. Tes A will further catalyze the formation of fatty acid through the dissociation of Acyl-ACP. The more fatty acid, the more pfadBA will be activated and more tesA protein will be produced. This will form a positive feedback loop and thus the fold change will become higher.
Reference
[1] Janßen, H. J., & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels, 7(1), 7.
[2] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 656
Illegal AgeI site found at 768 - 1000COMPATIBLE WITH RFC[1000]
| n/a | PfadBA-RBS-mRFP |