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Characterization of Existed Parts on Registry

1. CrtEBI Characterization

Biobrick BBa_K274100 is the lycopene-producing device submitted by team Cambridge 2009. It is used to produce lycopene which is red pigment can be detected by the naked eye.

However, we find that BBa_K274100 represented a significant leakage expression when exploited as a reporter gene and even bacteria bearing BBa_K274100 only could also represent significant color change, compared with the blank. We speculate that it’s because of a putative promoter upstream, resulting in the leaky expression of CrtEBI. In order to verify the speculation, we further characterize this biobrick. This biobrick was suffixed to the constitutive promoter BBa_J23103. On the other hand, we did something fantastic: The biobrick was suffixed to the terminator BBa_B0015. Terminator upstream was expected to reduce the basal level of lycopene production, thus to verify our speculation. Resulted two new biobricks are shown in Figure 1.

Figure 1 Biobricks we constructed to characterize biobrick BBa_K274100. The left construct denotes Constitutive promoter BBa_J23103-CrtEBI and on the right is an interesting biobrick—Terminator BBa_B0015-CrtEBI, namely a terminator was prefixed to CrtEBI, expected to reduce the leaky expression.

After the two biobricks was constructed, they were transformed to JM109, BL21 (DE3) and DH5α, respectively. Then we compared their color with the corresponding strains that bear plasmid with only CrtEBI as the insert, at different time intervals.

After cultivated at 37℃ for 9 hours, as shown in Figure 2, bacteria bearing BBa_J23103-CrtEBI and naked CrtEBI represented a light red color while bacteria bearing Terminator-CrtEBI and blank vector appeared white (Fig 2). We can’t collect bacteria of strain BL21 (DE3) at this time because it was slow-growing in this experiment for some reason.

Constitutive promoter BBa_J23103-CrtEBI

Terminator BBa_B0015-CrtEBI

Naked CrtEBI

Blank

Fig. 2 Color contrast after 9 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α).

After 12 hours’ incubation, strain BL21 (DE3) appeared white in all cases. Besides, constitutive promoter BBa_J23103-CrtEBI appeared red while CrtEBI still represented light red. The terminator-CrtEBI appeared light red in strain JM109 and still white in DH5α. The results are shown in Figure 3.
Constitutive promoter BBa_J23103-CrtEBI

Terminator BBa_B0015-CrtEBI

Naked CrtEBI

Blank

Fig 3. Color contrast after 12 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α).

After 15 hours, as was shown in Figure 4, the situation was the same as 12 hours. The only difference was that strain BL21 (DE3) appeared red color. BL21 (DE3) constitutive promoter BBa_J23103-CrtEBI appeared red while naked CrtEBI appeared light red. BL21 (DE3) terminator-CrtEBI represented white like the blank.