TABLE 2.
Strains, plasmids, and primers used in this studya
| Strain, plasmid, or primer | Relevant characteristic | Source or reference |
|---|---|---|
| Strain | ||
| L. crispatus ST1 | Used for the amplification of the anchor YSIRK sequence | ATCC |
| L. acidophilus ATCC 4356 | Host strain for fusion protein genomic integration | ATCC |
| E. coli BL21 (DE3) | Host strain for protein expression in Escherichia coli | Novagen |
| Plasmid | ||
| pTRKH3-ldhGFP | Cloning vector for protein expression in Lactobacillus | Addgene (19) |
| pLP401T | Cloning vector for protein expression in Lactobacillus | (21, 44) |
| pUC18 | Cloning vector in E. coli | NEB, Inc. |
| pET28a | Inducible expression vector in E. coli | Novagen |
| pWZ427 | pET28a-GFP-CD4 | This study |
| pWZ486 | pTRKH-Pldh-SP1-GFP-CD4-ANC, Ermr | This study |
| pWZ521 | pTRKH-Pldh-SP2-GFP | This study |
| pWZ528 | pUC18-Ermr | This study |
| pWZ531 | pUC18-Ermr-Upper-down (landing pad) | This study |
| pWZ535 | pUC18-Ermr-Pldh-SP1-GFP-CD4-ANC, Ampr, Ermr | This study |
| Primer | ||
| 001 | GCGGAATTCTGTTTTGAATTTTGTCATTGTCG | erm resistance gene, forward |
| 002 | GCGGAATTCTTAATTAATGAGACAGGTTTTAAGCAACTTGC | erm resistance gene, reverse |
| 037 | GCGCCTGCAGCTATTCTTCACGTTGTTTCCGTTTC | Long anchor of PrtP, reverse |
| 048 | GCGCGAATTCGCAGTCGACAAGCTTTTTAGTC | Pldh, forward |
| 057 | AATTCTCGAGTCCTGAGCCTTTGTATAGTTCATCCATG | GFP, reverse |
| 072 | GTATAATTATAGCACGACCTCTGATAAATATGAACATG | erm with mutated SacI site, forward |
| 073 | CATGTTCATATTTATCAGAGGTCGTGCTATAATTATAC | erm with mutated SacI site, reverse |
| 118 | GGCCGAGCTCAAGAAGGTTGTTTTAGGTAAGA | CD4, forward |
| 119 | GCTTTTCAAAAGGCTTCATCAGGAGGTAGTAAAGGAGAAG | CD4-GFP overlap, forward |
| 120 | GAAAAGTTCTTCTCCTTTACTACCTCCTGATGAAGCCTTTTGAAAAGC | CD4-GFP overlap, reverse |
| 141 | GCGACTCGAGGATAAGAAGACTTCGCTGC | Long anchor of PrtP, forward |
| 152 | CTAATAAAAAAGGAGACTTGACTTCCATGAAGCGACTTAAATTTTTAG | Pldh-SP1, forward |
| 153 | CTAAAAATTTAAGTCGCTTCATGAAGTCAAGTCTCCTTTTTTATTAG | Pldh-SP1, reverse |
| 154 | GCAGCAACCATAGAAAGCGGAGGTAGTAAAGGAGAAG | Pldh-SP1-GFP, forward |
| 155 | CTTCTCCTTTACTACCTCCGCTTTCTATGGTTGCTGC | Pldh-SP1-GFP, reverse |
| 164 | GGACGACGGGAACTACAAGAC | GFP, forward |
| 173 | GCGCCTGCAGTTACTCGAGTGATGAAGCCTTTTGAAAAGC | CD4, reverse |
| 184 | GCGCGTTAATTAATCCTTTAAACTCATCAAAAGCCAAATG | Upper pad, forward |
| 185 | GGCAGTTAATTAATCATTTCTTTTGATCAAAACACTTAC | Upper pad, reverse |
| 186 | GCGAAAGCTTGTTTAAACATTTTACTATCGCCAATG | Down pad, forward |
| 187 | GCGAAAGCTTGTTTAAACCTGCGGCTACTTGATTAGCTTTAG | Down pad, reverse |
| 194 | GTTAGGAAACCAAGCTCTGAC | Forward primer to test integration |
| 195 | GCCAGAATATGCTTGCGTCT | Reverse primer to test integration |
a
Ermr, erythromycin resistance gene; Ampr, ampicillin resistance gene; Pldh, promoter; SP, signal peptide; ANC, anchor domain; prtP, serine proteinase anchor domain.