Part:BBa_K2669003:Experience

These results were obtained with incorporated plasmid containing the composite part sequence in the IDT supplied backbone.

Transformation results

Figure 1: Transformation plate of colonies with the incorporated plasmid containing the IDT optimised original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the stability assay are circled.

Figure 2: Transformation plate of colonies with the incorporated plasmid containing the original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the assay are circled.

Stability assay

Stability assay through growth in liquid culture was performed with both inserts in the IDT supplied backbone. 1 mL of LB with ampicillin (25 ug/ml) was inoculated with one single colony in eppendorf tubes in 10 replicates for each part. In order to allow 10 generations of growth, a 1:1000 dilution was made of the overnight culture and incubated for 24 h. The colour comparison was done through visualization of centrifuged cultures and by analyzing the color intensity we could deduce the stability of the chromoprotein-encoding plasmid.

Figure 3: The result after 10 generations. The upper row represents 10 different culture pellets of the cells with the plasmid containing the IDT optimized sequence of AmilCP. The lower row represents the ones with the original native AmilCP (BBa_K592009) sequence incorporated in the plasmid.

Interpretation

Already after 10 generations it was clearly visible that the color intensity was better distributed in the cells containing the plasmid with the IDT optimized AmilCP sequence, since all of the culture pellets had a large color intensity while the cells with the plasmid containing the original AmilCP sequence had a larger variation in color intensity. This indicates that the stability of the cells containing the plasmid with the IDT optimized AmilCP sequence is greater than those with the original AmilCP sequence.

Table 1: Interpretation of color expression of the cells containing the original AmilCP sequence or the IDT optimized AmilCP sequence after 10 generations of growth. ++ represents strong color, + represents weak color and - represents no color.

Generation	Original AmilCP Colony 1	Original AmilCP Colony 2	Original AmilCP Colony 3	Original AmilCP Colony 4	Original AmilCP Colony 5	Original AmilCP Colony 6	Original AmilCP Colony 7	Original AmilCP Colony 8	Original AmilCP Colony 9	Original AmilCP Colony 10
10	++	+	++	++	+	+	++	++	++	+
Generation	IDT Optimized AmilCP Colony 1	IDT Optimized AmilCP Colony 2	IDT Optimized AmilCP Colony 3	IDT Optimized AmilCP Colony 4	IDT Optimized AmilCP Colony 5	IDT Optimized AmilCP Colony 6	IDT Optimized AmilCP Colony 7	IDT Optimized AmilCP Colony 8	IDT Optimized AmilCP Colony 9	IDT Optimized AmilCP Colony 10
10	++	++	++	++	++	++	++	++	++	++

Applications

AmilCP can be used as a quantitative reporter.

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