Table 1.
SureVector overlaps (N-terminal fusions).
Table 2.
SV devices required to re-create the DMRL biosynthetic pathway.
Fig 1.
Schematic of a seven SV part assembly into a GOI expressing device.
Different colored open rectangles highlight unique 30 bp overlaps between functional parts. Sequences represented by the two end rectangles (red) also overlap. The mixture of parts is treated with the SureVector enzyme assembly blend resulting in a device, represented by the closed circle that will transform, replicate and express a GOI in E.coli.
Fig 2.
Schematic Showing How Adjacent SV Parts are Assembled; Parts A and B Possess Homologous Ends.
Following denaturation and annealing, resulting free 3’ ends are extended, “flaps” digested and the two parts ligated.
Fig 3.
Collection of SV parts and assembly design.
A variety of parts and GOI’s can be assembled into many functional devices.
Fig 4.
a. SDS-PAGE gel of SV Nedd5 expression devices with different N-terminal tags. M = protein molecular weight marker; (-) = Uninduced sample; (+) Induced samples. SV denotes expression from SureVector assembled plasmids, ptac denotes expression from the commercially available ptac based plasmids. Green stars denote expressed N-terminal tagged Nedd5-fusion proteins. Fig 4b. SDS-PAGE Gel of SV Nedd5 Expression Devices with Different C-Terminal Tags. M = protein molecular weight marker; (-) = Uninduced samples; (+) = Induced samples. SV denotes expression from SureVector assembled plasmids, ptac denotes expression from the commercially available ptac based plasmids. Green stars denote expressed N-terminal tagged Nedd5-fusion proteins.
Fig 5.
The imidazole ring of GTP is hydrolytically removed by GTP Cyclohydrolase II (rxn. 1; ribA) yielding 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone-5’-phosphate (DARPP), formate and pyrophosphate. DARPP is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione-5’-phosphate (ARPP) by fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione reductase (rxns. 2; both ribD). Separately, ribulose-5-phosphate (R5P) is converted to L-3,4-dihydroxy-2-butanone-4-phosphate (DHBP) and formate by L-3,4-dihydroxy-2-butanone-4-phosphate synthase (rxn. 3; ribB). ARPP and DHPB are dephosphorylated and then condensed by DMRL synthase (rxn. 4; ribE) producing 6,7-dimethyl-8-ribityllumazine (DMRL).
Fig 6.
Co-transformation of two SV compatible devices containing all 4 rib biosynthetic genes results in DMRL synthesis.
Devices K6 and A7 (see Table 2) were co-transformed into Agilent BL21(Gold)DE3 E.coli and spread onto LB-kan-amp-IPTG plates. Resulting colonies were examined under unfiltered UV light. All resulting K6A7 systems contained four rib genes and produced DMRL as evidenced by fluorescent colonies with fluorescent halos. Control (device K5A5) through three rib genes (see Table 2) did not produce DMRL as no fluorescent halos were detected.
Table 3.
Combinations of Table 2 functional and control devices used to re-create the DMRL biosynthetic pathway system.
Fig 7.
Total quantities of DMRL synthesis from devices and systems.
Single colonies from devices and systems listed in Table 3 were purified by re-streaking onto fresh LB-kan-amp plates without IPTG. Three colonies from each plate were cultured in separate tubes containing 3 ml of LB-kan-amp. One hundred microliters of each culture were added to 4.9 ml of LB-kan-amp and incubated until OD600 reached between 0.3 and 0.5. IPTG was added to a final concentration 0.5 mM and incubation continued for three hours after which OD600 values were re-measured. Cultures were centrifuged to remove cells and OD409 values of supernatants obtained. OD409 values were normalized relative to OD600 values measured post IPTG addition and the resulting numbers compared. DMRL was synthesized exclusively by systems containing two devices each expressing two rib genes–systems K6A7, K7A6, K8A9 and K9A8.
Fig 8.
DMRL synthesis rates of systems K6A7, K7A6, K8A9, K9A8 and negative control K5A5 were obtained by inoculating single colonies into 5 ml of LB-kan-amp media followed by overnight incubation at 37°C with shaking at 250 rpm. One ml of these starter cultures was inoculated into separate 250 ml flasks each containing 49 ml of LB-kan-amp. Incubation at 37°C with shaking at 250 rpm continued until OD600 values reached between 0.3 and 0.5. IPTG was added to a final concentration of 0.5 mM and incubation continued. At regular intervals, 1.0 ml samples were retrieved from each culture and OD600 values measured. Samples were then centrifuged to remove cells and OD409 values of supernatants obtained. OD409 values were normalized by dividing by the OD600 values and these numbers plotted as a function of OD409 post-IPTG addition.