TABLE 1.
pAMY-em1 constructs and their pAD1-related genotypes
| Construct | Description | Genotype |
|---|---|---|
| pBCAEYP31 | Complete sequence of regulatory regionsa from traB (BspHI site) to the asa1 start codon (BspHI site) | traBCAE1orfY1 |
| pCAEYP31 | ΔtraB (PvuI/PacI fragment deleted from pBCAEYP31) | traCAE1orfY1 |
| pBAEYP31 | ΔtraC (PvuII fragment deleted from pBCAEYP31) | traBAE1orfY1 |
| pBEYP31 | ΔtraC/A (ca. 300-bp deletion within traA introduced into pBAEYP31) | traBCAE1orfY1 |
| pAEYP31 | Complete sequence of regulatory region from traA (HincII site) to the asa1 start codon (BspHI site) | traAE1orfY1 |
| pP0EYP31 | Complete sequence of regulatory region from P0 (NheI site) to the asa1 start codon (BspHI site) | traE1orfY1 |
| pP1/2YP31 | Complete sequence of regulatory region from P1/2 (NsiI site) to the asa1 start codon (BspHI site) | orfY1 |
| pAEP31 | orfY (NsiI-Sau3a fragment) deleted from pAEYP31 | traAE1orf1 |
| pAEY-RBSamy | Complete sequence of regulatory region from traA (HincII site) to orfY (Sau3a site): traAE1orfY; amyL controlled by its original RBS (RBSamy) | traAE1orfY |
| pP31 | Fragment from P3 (HpaII site) to the asa1 start codon (BspHI site): orf1 | orf1 |
| pEA/YP31 | pAEYP31 with inversion of a HincII-NsiI fragment covering traA through traE1 | traAE1(Inv)-orfY1 |
| pEA/P31 | orfY (NsiI-Sau3a fragment) deleted from pEA/YP31 | traAE1(Inv)-orf1 |
| pEA/RBSamy | HincII-NsiI fragment covering traA through traE1 inverted relative to amyL; amyL controlled by its original RBS (RBSamy) | traAE1(Inv) |
| pEP0/P31 | NdeI-NsiI fragment covering P3 through traE1 inverted relative to amyL; sequence from P3 (HpaII site) to the asa1 start codon (BspHI site) in original orientation | traE1(Inv)-orfY1 |
| pAEYP3(1) | pAEYP31 with a stop codon introduced into orf1 (1-nucleotide exchange creating a BamHI site near the orf1 start codon) | traAE1orfY-Δorf1 |
| pA(E)YP31 | pAEYP31 containing a −2 frameshift within traE1 | traA-ΔE1-orfY1 |
| pA(E)Y′-P31 | pA(E)YP31 with orfY 3′-end deleted (AciI-Sau3a fragment) | traA-ΔE1-orfYΔ3′-orf1 |
| pA(E)P31 | pA(E)YP31 lacking P1/2orfY (NsiI-Sau3a fragment) | traA-ΔE1-orf1 |
| pP0(E)P31 | pA(E)P31 with traA 3′-end deleted (NheI-HincII fragment) | ΔtraE1-orf1 |
| pAT1-′EP31 | pAT1-′EYP31 lacking P1/2orfY (NsiI-Sau3a fragment) | traA-E1Δ5′-orf1 |
| pP0T1-′EP31 | pAT1-′EP31 with traA 3′-end deleted (NheI-HincII fragment) | traE1Δ5′-orfY1 |
| pAE′-P2YP31 | 3′-end of traE1 and P1 promoter deleted from pAEYP31 (FspI-BspEI fragment) | traA-E1Δ3′-orfY1 |
| pAP0P1/2YP31 | pAEYP31 with traE1 deleted (XbaI-NsiI fragment) | traAorfY1 |
| pAP0P31 | pAP0P1/2YP31 lacking P1/2orfY (NsiI-Sau3a fragment) | traAorf1 |
| pP0P1/2YP31 | pP0EYP31 with traE1 deleted (XbaI-NsiI fragment) | orfY1 |
| pRBSamy | Control plasmid corresponding to the pAMY-em1/pWM401 cointegrate | |
| pPermRBSamy | Control plasmid corresponding to the pERM-ex1/pWM401 cointegrate (26) |
a
“Complete sequence of regulatory regions” indicates that the original sequence within the given fragment remained unchanged, with the following exceptions: (i) the NsiI site downstream of traE1 contains 8 additional bp derived from cloning vector pOK12 (TGCAGGCA), and (ii) sea1 encoding surface exclusion protein (36) is completely lacking and has been replaced by 8 bp derived from cloning vector pUC19 (TCTAGAGT).