Composite

Part:BBa_K2924020

Designed by: Mirko Kraus   Group: iGEM19_Duesseldorf   (2019-10-14)
Revision as of 17:30, 21 October 2019 by AndreasN (Talk | contribs) (→‎Usage and Biology)


Promoter AR with the chromoprotein amilCP

Long-chain fatty acid sensitive promoter PAR expressing amilCP. This part was used to characterize amilCP.

Usage and Biology

Fig.1: LB-Agar plate of Escherichia coli Top10F with the pBbB6c + amilCP plasmid. The amilCP makes the colonies blue.
Fig. 2: Regulation of fatty acid metabolism by the transcriptional factor FadR. FadR recognizes its cognate binding site (white), thereby repressing transcription. Upon binding of an Acyl-CoA to FadR, the promoter region is freed, enabling gene expression.

This year the iGEM team from Düsseldorf 2019 was working on the characterization of amilCP, a blue chromoprotein from the Acropora millepora (BBa_K592009) for the use as a reporter gene for a biosensor. For the characterization the synthetic PAR from a previous publication 1 was used and so the biosensor is sensitive for long chain fatty acids (LCFA). The plan was to implement a chromoprotein as a reporter gene, thus enabling to every laboratory to measure a biosensor without fluorescence applications - in contrast, most laboratories have access to a photometer. The chromoprotein amilCP can be observed with the naked eye, in addition to the measurement method by absorption at the wavelength 588 nm for example with a plate reader. Therefore, this option is available for everyone.

Chromoproteins are a good alternative to fluorescent proteins like red fluorescent protein (RFP). The amilCP protein has an absorption maximum at 588 nm and the blue color can be seen with naked eye. Chromoproteins have the benefit that they can measured by absorption and thus can be used in every laboratory.

The production of long-chain fatty acids (LCFA) is regulated by the transcription factor FadR. This transcription factor can bind to Acyl-CoA which leads to the release of FadR from its cognate operator sequence, thereby increasing gene expression (as shown in Fig. 2).



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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The constructs were cloned into a variant of the pBbB6c 2 medium copy number backbone which lacked the lac-promoter, with the restriction sites EcoRI and XhoI. This backbone has the antibiotic resistance chloramphenicol and the origin of replication (ori) pBBR1. The Escherichia coli strain Top10F was transformed with this biosensor plasmid, as seen in Figure 3.

Fig.3: Promoter AR with reporter gene amilCP in a pBb backbone. The backbone has a chloramphenicol resistance and a medium copy ori p15A. EcoRI and XbaI were used as restriction enzymes.

The transformation of the organism was validated by colony PCR. In some cases, successful transformation could quickly be detected by the different colors of the colonies on the plate and as a cell pellet.

The positive clones were used for experiments and were grown in LB-medium over night. The fatty acid stocks for the experiments were prepared. All of the fatty acids were dissolved in ethanol with the exception of butyric acid, which was dissolved in water. The stock solutions of the fatty acids are shown in Tab.1.
Tab.1: Stock solutions for different fatty acids

Fatty acid Chain length MW [g/mol] Solvent Solubility Stock concentration
Butyric acid C4:0 88.11 Water 60 g/L 200 mM
Capric acid C10:0 172.26 Ethanol 30 g/L 100 mM
Lauric acid C12:0 200.32 Ethanol 20 g/L 100 mM
Myristic acid C14:0 228.37 Ethanol 15 g/L 75 mM
Palmitic acid C16:0 256.42 Ethanol 20 g/L 75 mM
Stearic acid C18:0 284.48 Ethanol 20 g/L 50 mM
Oleic acid C18:1 282.46 Ethanol 100 g/L 200 mM

The absorption of the overnight cultures were measured and the cultures were used to inoculate fresh LB medium to an OD600 of 0.05. The cultures were induced with different concentrations of fatty acids. First preliminary tests were carried out with palmitic acid at final concentrations of 0.4 mM, 1 mM and a control without fatty acid. For a better dissolving of the hydrophobic fatty acids in LB-medium, Tergitol which was 0.5% of the whole volume, was added to the medium. The induced samples were transferred on a 24 well plate and the plate was incubated overnight in a 37°C incubator, which shaked the cultures with 250 rpm. After nearly 16 hours, the samples were taken out of the incubator and distributed on a 96 well plate in 200 µL aliquots. To account for biological heterogeneity and technical errors three biological replicates were measured in three technical replicates each. An empty vector control (EVC) was also supplemented with fatty acids at different concentrations and measured. All experimental details are listed in Tab. 2.
Tab. 2: List of all part improvement experiments

Construct Fatty acid Concentrations
PAR:amilCP Lauric acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:amilCP Myristic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:amilCP Palmitic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:amilCP Stearic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Lauric acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Myristic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Palmitic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Stearic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM

Fig.2: Response of PAR+amilCP (blue) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.

The experiments with the blue chromoprotein amilCP ((BBa_K592009) 1 showed that the production of the chromoprotein is increased by a higher concentration of fatty acids in the medium. The best result was achieved with the fatty acid stearic acid. The biosensor also worked for the chain lengths from C14:0 and C16:0. Here, the EVC is lower than the samples with amilCP. By adding lauric acid (C12:0) to the culture medium, the production of amilCP increased, but the EVC also increased in a similar manner close to amilCP, so it is not certain if this result can be used for distinct conclusion.

To summarise the experiments, the created biosensors works for the fatty acids myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0) with every reporter gene. When using lauric acid (C12:0), the results become more inaccurate. A dose response can still be observed, but it is not as strong or linear as with the other fatty acids.











References

1: https://parts.igem.org/Part:BBa_K592009

2: Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. ”BglBrick vectors and datasheets: A synthetic biology platform for gene expression.” J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410

3: Fuzhong Zhang, James M Carothers, Jay D Keasling. “Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids” Nature Biotechnology volume 30, pages 354–359 (2012)

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