Difference between revisions of "Team:UPF CRG Barcelona/Results"

RESULTS

Overview

• When fadL and fadD are overexpressed LCFA uptake increases resulting in a higher growth if LCFA are the only carbon source available.
• pFadBA-luxR-pLux lactone inducible LCFA biosensor can amplify the signal of pfadBA promoter-controlled gene expression.
• pFadBA-cI-pRM LCFA biosensor reduces a lot the baseline expression of genes directly regulated by the pfadBA promoter.
• pAR promoter significantly improves pfadBA function as an off/on switch dependent on the presence of LCFA.
• We used MAGE recombineering to generate a strain missing fadR, in a effort to integrate intake capabilities in to the genome.
• We applied the PfadBA based biosensor to improve screening of recombineered fadR colonies.

Overexpression results

In order to study the functionality of our system we characterized the constructs both in palmitic acid (PA) and oleic acid (OA). Bacteria was grown for 3 days in M9 minimal medium with the respective fatty acid, to ensure that cells were using solely LCFA as a carbon source . Additionally, the supernatant was also collected and LCFA concentration was determined using cupric-acetate colorimetric (see methods) technique at OD715nm. The same growth assay was performed in LB to infer cell growth in enriched media.

Different levels of fad genes expression were achieved through the use of different strains. Genetic constructs consisting of either FadD or FadL downstream of the tetR repressible promoter were transformed in Top10 (DH5-alpha) and Zn1 E. coli strains . Top 10 constitutively synthesizes tetR, which results in a continuous expression of our construct. Zn1 does not express tetR, thus meaning that our genetic constructs will not be induced in this strain. This induction can be modulated by adding anhydrotetracycline (ATc) in the medium. We created a cell library expressing the fadD and fadL proteins:

Cell	Parts Registry	Construct	Expression	Cellular type
Constitutive FadD	BBa_K2581009	T14_ptet_32_FadD	constitutive	TOP10
Inducible FadD	BBa_K2581009	T14_ptet_32_FadD	tuneable	ZN1
Constitutive FadL	BBa_K2581010	T14_ptet_32_FadL	constitutive	TOP10
Inducible FadD	BBa_K2581010	T14_ptet_32_FadL	tuneable	ZN1
Constitutive Reporter	BBa_K2581010	T14_ptet_32_RFP	constitutive	TOP10

Table 1 | Summary of the parts and experimental conditions used for this section

FadD overexpression increases growth when either PA or OA are the only carbon source.

We analyzed the behavior of the constitutive and inducible FadD cell lines with M9 minimal media supplemented with either PA or OA. Constitutive reporter cell line was used as a control. OD measurements were performed at OD 600 nm as an indicative for cell growth for 72 hours.

Our results show that, when fadD is overexpressed, bacterial growth increases both in 0.4 mM PA (Fig 1A) and 2 mM PA (Fig 1B) in relation to control. Moreover, non-induced FadD cells show a lower increase of bacterial growth, compared to the control. On the other hand, when tested with OA medium (Fig 1C and Fig 1D), the difference in growth is higher than in PA medium.

FadD Overexpression in Minimal Medium