Table 1 |
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Oligonucleotides for QRT-PCR and genetic manipulations. |
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Primers for QRT-PCR |
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Name |
Purpose |
Location |
Sequence (5' to 3') |
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MA0326 |
spacer #2 |
CCTGGTTGAGGTTAGCGTTGA |
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PCR of spacer #1 |
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MA0327 |
outside CRISPR |
AATTCGGTGGCCAGTTGTTC |
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MA0328 |
sense strand |
CAGGAAGCCACCAAGGAT |
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PCR of hisS Pcar_1041 |
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MA0329 |
antisense strand |
TGGGAGCCGAGTTGATTG |
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MA0441 |
sense strand |
CAAACTGATTGCCGTTCCTT |
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PCR of hisZ GSU3307 |
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MA0442 |
antisense strand |
AGGCCGATGAGTTCTACGC |
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Primers for construction of hisS transgenic strain MA159 |
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Name |
Purpose |
Sequence (5' to 3') |
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MA0330 |
TGACATCTCGCTGGACCGGG |
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PCR on 5' side of hisS GSU1659 |
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MA0331 |
CTATGCTAGCACTAGTTTGTAATCATGAACGTACCTACTC CTTTAATTG |
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MA0332 |
GTACGTTCATGATTACAAACTAGTGCTAGCATAGCAATAC CTGCATTG |
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PCR on 3' side of hisS GSU1659 |
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MA0333 |
AGTCCATTCCTCCTGTGG |
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MA0334 |
AAGGGATCTATCATGAGCATATCAGGCATTAAGGG |
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PCR of hisS Pcar_1041 |
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MA0335 |
GCGCGGCGCGACTAGTTTCCTCGTGTCTTTTCC |
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MA0052 |
TGCATATGGCTCTAGAATAACTTCGTATAGC |
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gentamicin marker |
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MA0053 |
TCGATAAGCTTCTAGAATAACTTCGTATAATG |
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Oligonucleotides for construction of chimeric CRISPR expression plasmid pMA35 |
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Name |
Purpose |
Sequence (5' to 3') |
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MA0269 |
ACATGTCACTGCCCGCTTTCCAGTC |
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PCR of lacI- taclacUV5 promoter |
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MA0270 |
GCATGCGTGTGAAATTGTTATCCGC |
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MA0429 |
AATTCGGTTCATCCCCGCGCATGCGGGGAACACATACAT GAGGGCAAACGCCTTTTGGCCGGCGGCGGTTCATCCCCG CGCATGCGGGGAACACG |
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synthetic CRISPR of spacer #1 |
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MA0430 |
GATCCGTGTTCCCCGCATGCGCGGGGATGAACCGCCGCC GGCCAAAAGGCGTTTGCCCTCATGTATGTGTTCCCCGCAT GCGCGGGGATGAACCG |
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MA36R |
sequencing |
CGACATCATAACGGTTC |
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Note: Within the sequence of the chimeric CRISPR, a single base pair (underlined) has been duplicated in plasmid pMA35-!, at the exact centre of spacer #1. |
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Aklujkar and Lovley BMC Evolutionary Biology 2010 10:230 doi:10.1186/1471-2148-10-230 |
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