Coding

Part:BBa_K2669002:Experience

Designed by: Elin Ramstrm   Group: iGEM18_Uppsala   (2018-10-02)
Revision as of 16:08, 7 October 2018 by MatildaBrink (Talk | contribs) (→‎Stability assay)

Note that the results was gotten with the plasmid design represented in the composite part: IDT optimised AmilCP sequence

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 colonies 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 optimised AmilCP sequence, since all of the colonies 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 optimised AmilCP sequence is greater than those with the original AmilCP sequence.

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 +++ ++ +++ +++ ++ ++ +++ +++ +++ +

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

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 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

Table 2: Interpretation of color expression of the IDT optimised AmilCP sequence after 10 generations of growth. +++ represents strong color, ++ represents moderate color, + represents weak color and - represents no color.

Applications

AmilCP can be used as a non quantitative reporter.

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