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Contents 1 Results 1.1 E. coli DH5a is sensitive to Catechol exposure 1.2 Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate 1.3 Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling 1.4 Recombinantly expressed microcompartments assemble in vivo

Results

E. coli DH5a is sensitive to Catechol exposure

To measure the effect of our pathway manipulations on the viability of E.coli exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of E.coli (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in E.coli and P. putida have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered E.coli strains containing various configurations of the pathway.

A

B

Figure 1: A. Growth of E.coli DH5a on LB plates (1x10^-5 dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of E. coli DH5a exposed to catechol over a six hour time course.

Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate

Justification and summary of flux balance results which are described in more detail in the modeling section but highlighted here. The important point to make is that this is good news because, if true, it means that acceleration of the native pathway in the target organism P. putida would likely result in bacteria capable of outcompeting their normal counterparts, reducing the need to re-apply.

Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling

Kris, you need a blurb here on this early Cell++ result.

Recombinantly expressed microcompartments assemble in vivo

Kris your EM goes here with the appropriate description.