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Characterization and improvement

XJTLU-CHINA 2016 iGEM team build a mutagenesis library of A1 using degenerated primers. One set of degenerated primers were designed to mutate the poly-A-loop structure of A1 in order to tight the hairpin structure and the other set of primers were designed to modify Anti-SD of the ribothermometers. The primer sequences used were:

5'-ACAGAGAACATACTAGAGNNNNNTAAAAAAAAAAAGTACTAAGGAGTACTAG-3'

5'-AGAGAACATACTAGAGCTCNNNNNNNNNNNNNNGTACTAAGGAGTACTAG-3'

The mutagenesis pool of A1 was downstream linked to an mRFP reporter gene (see Fig.2) in order to determine the protein expression efficiently. Total 96 colonies were picked after the transformation of mutagenesis library and initially screened using 96 wells plates at 26 ℃ and 37 ℃. Ten colonies with the viable difference in color were selected for further analysis.

Figure 2. The mRFP expression construct that used for the screening of effective ribothermometers

These colonies were seeded in 150ml flasks that contain 20ml LB culture for 12 hrs. The fluorescent was then measured at an excited
length of 580nm and emitted length of 607nm. OD 600 of each sample was measured at the same time.

Figure 3. The difference in performance of sensing temperature of 10 A1 mutants.

All 10 obtained mutants were sent for sequencing and the result is included in the table below.

Table 1. Sequencing results of 10 mutants with broad-spectrum of sensitivity in respond to temperature

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