This protocol contains six steps:
        1. Construction of standard part to express reporter protein
        2. Mutation position(s) selection
        3. Degenerated primers design
        4. PCR amplification of target plasmid
        5. PCR product ligation and transformation
        6. RBS activity measurement

1. Construction of standard part to express reporter protein.

    The RBS part (B0034) was selected for RBS point mutation to generate RBS library. This RBS is a component of the pSB-I13522, which is composed of a Ptet promoter, a common RBS (B0034), a reporter gene (GFP gene) and dual Terminators .

Fig. 1: The composition of parts of I13522.

     The part pSB_I13522 was inserted into the low copy number backbone pSB3T5 to express GFP protein (Fig. 32).

Fig. 2: The part pSB_I13522 was inserted into the low copy number backbone pSB3T5.

2. Mutation position(s) selection

    The conserved sequence of RBS is AGGAGAA (Red label in Fig.13). To prevent the disruption of RBS basic functions, we chose the 2 base pair next to the conserved sequence of RBS for point mutation (Blue label in Fig.13).

Fig. 3: The sequence of RBS part BBa_B0034. The conserved sequence is marked in red and the point mutation sites in are marked in blue.

3. Degenerated primers design

   The primers containing two base pair mutations were designed as follows (Fig.4). The letter N designate sites containing ATCG bases.

      Forward:5'CTAGAGAAAGAGGAGAANNACTAGATGCGTAAAGG3'
      Reverse:5'CCTTTACGCATCTAGTNNTTCTCCTCTTTCTCTAG3'

Fig. 4: The degenerated primers were designed to amplify the RBS part BBa_B0034.

4. Using PCR to amplify the target plasmid

    PCR reaction mixture shown in Table 1. Note, it important to use KOD-plus polymerase. Because DNA polymerase from Termococcus kodakaraensis KOD is one of the most efficient, thermostable PCR enzymes exhibiting higher accuracy and elongation velocity in comparison to other available commercial DNA polymerase, we recommend its use.

 PCR condition Table 2

5. PCR product ligation and transformation

    The components of this ligation step are listed in Table 3. When the ligation mixture is prepared, move on to:

        (1) Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
        (2) Incubate the mixture for 8 hour at 16°C.
        (3) Use the mixture for transformation.

Table 3: The components of the ligation mixture.

    Ligation product is transformed in ECOSTM competent cells.
    Steps:
      1. Thaw competent cells in the room temperature water bath with circulating water or hold the tube under running tap water for ~20 seconds until 1/3~1/2 volume is thawed.

      2. Add DNA (pre-chilled on ice, volume should be ≦ 5% of competent cells) immediately. Vortex for 1 second or tap the tube with finger to mix well.

      3. Heat-shock the cells in the pre-warmed 42℃ water bath for 30~45 seconds.

      4. Plate the cells using plating beads onto a pre-chilled (4℃) and dried antibiotic-selected LB agar plate.

      5. Incubate the plates at 37℃.

6. RBS activity measurement

    Bacterial colonies were selected and cultured in M9 medium (with glucose). When bacteria grew to steady-state, 5μL bacterial medium was transferred into a new tube with 5ml M9 medium. Approximately 4-hours after incubation in shaker, the O.D. ratio of the bacterial medium should increase to 0.1, and the sample was measured for green fluorescence activity. All fluorescence measurements were performed on a flow cytometer equipped with a 488 nm argon excitation laser and a 515–545 nm emissions filter.

Fig.5: Green fluorescence intensity of the mutated RBS BBa_B0034. Mutated RBS translational activity from the high (left) to the low state (right).

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    For each sample, 100,000 events were collected. A small gate in the side-scatter and forward-scatter space was chosen to reduce the variations in cell size and help remove noise in fluorescence measurements. Fluorescence intensities were converted to molecules of equivalent fluoresces in based on daily measurements of SPHERO Rainbow Calibration Particles. We selected forty different colonies with one wild type colony as our samples and control (the green one in Fig. 6). The final data of green fluorescence activity shows only thirty-eight different data sets as two were later on excluded.

Fig.6: Green fluorescence intensity of colonies with the mutated RBS BBa_B0034. The green one is the wild type colony.

Fig.7: The green fluorescence intensity of colonies with the mutated RBS BBa_B0034. The mutated bases are designated on the bottom.

Fig.8: The green fluorescence intensity of colonies with the mutated RBS BBa_B0034 was measured for seven hours. Mutated bases are shown to the right

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