Team:SydneyUni Australia/Project/Design

dhlB Screening:
We expect a band at ~800bp for an amplification of dhlB, the gene in our pathway responsible for the degradation of chloroacetate. We don’t see it any of the 87 clones, which corresponds to our phenotypic assays of the clones, where few of the clones released many Cl- ions from the chloroacetate substrate.

Plasmid Digests
We did a second plasmid prep of our clones because the first weren’t very clear. Desmond figured out that by digesting the plasmids with EcoRV we ought to see a single 2kb band if pSB has closed on itself. We don't see anything like this in our clones, but we know from the rfp that this works (pSB-rfp is about 3kb, and contains a single EcoRV site in the pSB backbone). If the plasmid contains our gBlocks (or at least iGEMBLOCK 1 with aldA), then we expect to see two bands - one at 1kb, a second at 5 or 6kb (depending whether we're looking at p450 or the adh plasmid). We don't see this either. We reckon that in most of the plasmids, some gBlocks have assembled in pSB1C3 but not iGEMBLOCK 1. In one of the clones (64) we see a band at 1kb but nothing else, so maybe iGEMBLOCK1 assembled in pSB, but not other gBlocks.

Interestingly, rfp looks spot-on while our results are similar to that in the last plasmid prep (everything slightly higher than 3kb, except 64). With rfp we expect a single band at 3kb (pSB is 2kb plus the rfp 'part' is 1 kb, and the whole construct contains a single EcoRV site in pSB). Notably, 64 was one of the small p450 colonies we patched from Gibson Assembly Product transformations.

GA Product PCR Assembly
Ap, Bp, Cp are from the p450 pathway, using our GA reaction product as template. Aa, Ba and Ca are from the adh pathway. It looks like we can’t amplify what we want, or that it doesn’t exist in the GA reaction product.

PCR Junction Screening of AB22

We cloned the PCR fragments into pSB1C3 and transformed the ligation product. We were greatly assisted by ligating into a BBa_J04450, extracted from the linearised plasmid pSB1C3 in the Distribution Kit, which provided a neat red-white screen.

Colourmetric assay of chloride release from dhlB activity on chloroacetate (in blue) and dhlA activity on 1,2-Dichloroethane (1,2-DCA, in red). Standard curve generated with 0.0, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0mM NaCl in KP buffer. TOP10 E.coli cells were harvested at OD600=0.4, pelleted and washed three times in KP buffer. Cells were resuspended in 2mM chloroacetate or 1,2-DCA and incubated for 16hrs at 37°C and 200rpm. Cells were then pelleted and assayed using the chloride assay and the absorbance at 460nm read. Data for each condition is in triplicate (standard deviation <0.26 Cl- (mM)).

Chloride release from dhlB activity on chloroacetate (in blue) and dhlA activity on 1,2-Dichloroethane (1,2-DCA, in red). Negative Control was BBa_K1115008, the promoterless dhlB-dhlA coding region, BBa_K1115009 is dhlB-dhlA constitutively expressed by PCat (BBa_I14033]), BBa_K1115009 is constitutively expressed by PTet (BBa_R0040], and the positive control is the Coleman lab pUC19 house plasmid expressing dhlB-dhlA with the same RBS.