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From 2012.igem.org

Communication Bacteria<->Xenopus

Overview

Engineering Xenopus embryos with AID system raised the question of how will we deliver auxin to the embryonic cells? One idea was to use bacteria as a delivery machine in order to create a communication between two engineered organisms.

Steps

Our idea was to use previous biobricks from Imperial College 2011 BBa_K515100. Indeed, they managed to express in Escherichia coli the genes encoding the IAA-producing pathway from Pseudomonas savastanoi. Besides, we constructed a plasmid with a reporter (mRFP) as a control to monitor the auxin production.

Figure 1: Enginereed bacteria delivery to tadpole

Embryos were placed in medium containing MMR and DH5a bacteria with either BBa_K515100 or a reporter (mRFP), as shown below:

Figure 2: Fluorescence observations with different Bacteria concentrations



Figure 3: Fluorescence observations by day



Figure 4: Proof of principle

The results show that, except for the control, all tadpoles are fluorescent. The Fig.2 shows also that the fluorescence occurs mainly in the intestinal tract. The death rate during the experiments is close to 0%.
We've performed the same using the Imperial College 2011 plasmid; the tadpoles didn't show any sign of auxin intolerance.

Auxin detection

To detect whether auxins produced by bacteria diffuse in tadpole's gut and pass through membrane we conducted a serie of tests: Salkowski assay, High Pressure Liquid Chromatography (HPLC), Mass Spectrometry (MS).

1)Auxin standard






2)Control- head


3)Control- tail


4)Tadpole incubated with IAA producing bacteria- head




5)Tadpole incubated with IAA producing bacteria- tail

Conclusion

The fluorescence which we observe in tadpole's gut is derived from DH5a bacteria containing either BBa_K515100 or a reporter (mRFP). It proves that Xenopus embryos are capable to uptake bacteria and create a synthetic ecosystem. It is very interesting from the chassis-to-chassis communication point of view, because it is a new way of delivering specific molecules (encoded by bacteria) to Xenopus.For our project, using the bacteria as a new auxin delivering system represents a good alternative to the production devices 1 and 2, but only if auxin can migrate to the circulatory system .

HPLC and MS experiments were conducted to find out whether auxin produced by bacteria can be found in tadpole's tissues. Absence of IAA peak in both chromatograms and spectres can be explained by several hypothesis: Concentration of produced auxin was too low to be detected, samples were too diluted, auxin was degraded by tadpole or conditions in tadpole's gut are not optimal for bacterial metabolism so they don't produce auxins.