Table I. Cloning efficiency of every round of MISSA for pABA or pABA-oriT.
| Donor Straina | Expression Cassette | Recipient Strainb | Cm(S)/Gm(S)c | PCR(+)d | Cloning Efficiencye |
| % | |||||
| 1. pL-TM220 | KACsB/KA-TM220 | 20/20 (100%) | 16/20 (80%) | 80 | |
| 2. pR-NCED3 | pRD29A-NCED3-tNOS | KACsB/KA-TM220-NCED3 | 20/20 (100%) | 17/20 (85%) | 85 |
| 3. pL-TM1 | KACsB/KA-NCED3-TM1 | 20/20 (100%) | 8/10 (80%) | 80 | |
| 4. pR-tOCS | pRD29A-ABAR-tOCS | KACsB/KA-TM1-tOCS | 20/20 (100%) | 8/10 (80%) | 80 |
| 5. pL-ABAR | KACsB/KA-tOCS-ABAR | 20/20 (100%) | 4/10 (40%) | 40 | |
| 6. pR-TM2 | KACsB/KA-ABAR-TM2 | 17/20 (85%) | 4/4 (100%) | 85 | |
| 7. pL-CBF3 | pRD29A-CBF3-tNOS | KACsB/KA-TM2-CBF3 | 20/20 (100%) | 3/4 (75%) | 75 |
| 8. pR-LOS5 | pSuper-LOS5-tNOS | KACsB/KA-CBF3-LOS5 | 20/20 (100%) | 2/4 (50%) | 50 |
| 9. pL-ICE1 | pSuper-ICE1-tNOS | KACsB/KA-LOS5-ICE1 | 20/20 (100%) | 3/4 (75%) | 75 |
| 10. pRG-TM1 | KAGsB/KAG-IC1-TM1 | 20/20 (100%) | 1/4 (25%) | 25 | |
| 11. pLC-BAR | p35S-BAR-t35S | KACGsB/KAC-TM1-BAR | 13/20 (65%) | 4/4 (100%) | 65 |
| 12. pRG-GUS | p35S-GUS-tNOS | KACGsB/KAG-BAR-GUS | 5/20 (25%) | 4/4 (100%) | 25 |
| 13. pLC-TM2 | KACGsB/KAC-GUS-TM2 | 15/20 (75%) | 4/4 (100%) | 75 | |
| 14. pRG-HYG | p35S-HYG-t35S | KACGsB/KAG-TM2-HYG | 17/20 (85%) | 2/4 (50%) | 43 |
| 15. pL-TM220 | KACGsB/K-pABA | 8/20 (40%) | 4/4 (100%) | 40 | |
| 15. pL-oriT | KACGsB/KA-pABA-oriT | 16/20 (80%) | 4/4 (100%) | 80 |
The numbers before the donor strains correspond to the numbers of rounds of MISSA recombination. Regulations for the designation of the donor and the recipient strains are the same as those in Supplemental Table S2.
The randomly selected final clones of each round of MISSA were counterselected by streaking them on LB agar plates supplemented with chloramphenicol (Cm) and/or gentamycin (Gm). The clones sensitive (S) and resistant to Cm and/or Gm were counted separately, and the percentages of the Cm(S) or Gm(S) clones are indicated in parentheses.
The Cm- and/or Gm-sensitive clones were submitted for colony PCR identification. Those clones that were PCR positive for all the different DNA fragments tested were counted as appropriate ones and indicated as PCR(+). The total numbers of tested clones are indicated in the denominators of the fractions. The percentages of the PCR(+) clones are indicated in parentheses.
The cloning efficiencies were calculated by multiplying the percentage of Cm/Gm-sensitive clones by the percentage of the PCR(+) clones. The average cloning efficiency was calculated to be 62% (for pABA) or 64% (for pABA-oriT).