Table 1.
E. coli strains, plasmids and oligonucleotides used.
| Name | Relevant genotype | References |
|---|---|---|
| Strains | ||
| DH10B | F− mcrA (crmrr-hsdRMS-mcrBC) (r-hsdRMS-mcrBC) and oligonucleotide139 Δ(ara, leu)7697 galU galK alrpsL nupG | Life technologies (Carlsbad, CA) |
| BL21 Star (DE3) | F− ompT hsdSB (rB− mB−) gal dcm rne131 (DE3) | Life technologies (Carlsbad, CA) |
| JW2637-4 | F–, Δ(araD-araB)567, ΔlacZ4787(:rrnB-3), λ–, ΔgabT743:kan, rph-1, Δ(rhaD-rhaB)568, hsdR514 | CGSC #11775 |
| JW0036-1 | F–, Δ(araD-araB)567, ΔlacZ4787(:rrnB-3), λ–, ΔCaiC750:kan, rph-1, Δ(rhaD-rhaB)568, hsdR514 | CGSC #8352 |
| Plasmids | ||
| pDNA2.0-ORF27 | pUC ori, KanR, ORF27 with Biobrick overhang | DNA 2.0 (Menlo Park, CA) |
| pBbE2C- RFP | ColE ori, CmR, tetR, RFP | (2011) |
| pBbE2C-ORF27 | ColE ori, CmR, tetR, ORF27 | This study |
| pBbE2C-CaiC | ColE ori, CmR, tetR, CaiC | This study |
| pBbS2C-ORF27 | SC101 ori, CmR, tetR, ORF27 | This study |
| pET28b | pBR322 ori, KanR, lacI, MCS after PT7 | EMD Millipore (Billerica, MA) |
| pET28a-MBP | pBR322 ori, KanR, lacI, MCS after MBP fusion driven by PT7 | EMD Millipore (Billerica, MA) |
| pET28a-MBP-ORF27 | pET28a-MBP with ORF27 inserted into NdeI and XhoI | This study |
| pBbE2C-MBP-ORF27 | ColE ori, CmR, tetR, MBP-ORF27 | This study |
| pBbA5a-GadB | p15A ori, AmpR,Placuv5, GadB | This study |
| pBbA5a-GadB_ΔHT | p15A ori, AmpR, Placuv5, GadB_ΔHT | This study |
| pBbA5a-GadB_Δ1-14 | p15A ori, AmpR, Placuv5, GadB_ Δ1-14 | This study |
| pBbA5a-GadB_Δ1-14, ΔHT | p15A ori, AmpR, Placuv5, GadB_ Δ1-14, ΔHT | This study |
| pBbA7a-RFP | p15A ori, AmpR,PT7, RFP | T.S. Lee (2011) |
| pBbA7a-GadB | p15A ori, AmpR,PT7, GadB | This study |
| pBbA7a-GadB_ΔHT | p15A ori, AmpR,PT7, GadB_ΔHT | This study |
| pBbA7a-GadB_Δ1-14 | p15A ori, AmpR,PT7, GadB_ Δ1-14 | This study |
| pBbA7a-GadB_Δ1-14_ΔHT | p15A ori, AmpR,PT7, GadB_ Δ1-14_ΔHT | This study |
| Oligonucleotides | 5′→ 3′ Sequence, restriction site underlied, synthesized by Integrated DNA technologies, Inc (Coralville, IA) | Target gene |
| JZ_MBP-ORF27_f | GCGCGgaattcaaaagatcttttaagaaggagatatacatatgggcagcagccatcatca | MBP-ORF27 |
| JZ_MBP-ORF27_r | GCGCGctcgagtttggatccTCATTCTGCCGCCATACGGG | MBP-ORF27 |
| JZ_GadB_f | gcgcgCATATGgataagaagcaagtaacg | GadB, GadB_ΔHT, |
| JZ_GadB_r | gcgcgGGATCCTTAtcaggtatgtttaaagctgtt | GadB, GadB_Δ1-14 |
| JZ_GadBΔ1-14_f | gcgcgCATATGGGTTCACGTTTTGGTGCGA | GadB_Δ1-14, GadB_Δ1-14_ΔHT |
| JZ_GadBΔHT_r | gcgcgGGATCCTTAtcatttaaagctgttctgttggg | GadB_Δ1-14_ΔHT GadB_ΔHT |
| JZ_CaiC_f | GCGCgaattcaaaagatcttttaagaaggagatatacatATGGATAGAGGTGCAATGGAT | CaiC |
| JZ_CaiC_r | GCGCG ctcgagtttggatccTTATTTCAGATTCTTTCTAATTATTTTCCCC | CaiC |
| Strains | Plasmids | Host |
| JZ-171 | pBbE2C-ORF27 | JW2637-4 |
| JZ-172 | pBbE2C-RFP | JW2637-4 |
| JZ-298 | pBbS2C-ORF27 | JW2637-4 |
| JZ-299 | pBbE2C-MBP-ORF27 | JW2637-4 |
| JZ-336 | pBbE2C-MBP-ORF27+pBbA5a-GadB WT | BL21 Star (DE3) |
| JZ-338 | pBbE2C-MBP-ORF27+pBbA5a-GadB_ΔHT | BL21 Star (DE3) |
| JZ-339 | pBbE2C-MBP-ORF27+pBbA5a-GadB_Δ1-14 | BL21 Star (DE3) |
| JZ-340 | pBbE2C-MBP-ORF27+pBbA5a-GadB_Δ1-14, ΔHT | BL21 Star (DE3) |
| JZ-342 | pET28a-MBP-ORF27+pBbA7a-GadB WT | BL21 Star (DE3) |
| JZ-344 | pET28a-MBP-ORF27+pBbA7a-GadB_ΔHT | BL21 Star (DE3) |
| JZ-345 | pET28a-MBP-ORF27+pBbA7a-GadB_Δ1-14 | BL21 Star (DE3) |
| JZ-346 | pET28a-MBP-ORF27+pBbA7a-GadB_Δ1-14, ΔHT | BL21 Star (DE3) |
| JZ-348 | pBbE2C-MBP-ORF27+pBbA7a-GadB WT | BL21 Star (DE3) |
| JZ-350 | pBbE2C-MBP-ORF27+pBbA7a-GadB_ΔHT | BL21 Star (DE3) |
| JZ-351 | pBbE2C-MBP-ORF27+pBbA7a-GadB_Δ1-14 | BL21 Star (DE3) |
| JZ-352 | pBbE2C-MBP-ORF27+pBbA7a-GadB_Δ1-14, ΔHT | BL21 Star (DE3) |
| JZ-370 | pBbE2C-CaiC | JW2637-4 |
| JZ-386 | pET28b-N-CaiC+pBbA7a-GadB_ΔHT | BL21 Star (DE3) |
| JZ-393 | pBbE2C-RFP | JW0036-1 |