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The plasmids for quartromicin M5 (KS-AT-ACP), M3 ACP and M4 ACP were produced by Dr. Orestis Lazos (Challis group) using fosmid libraries previously produced by Malek Zerikly from the following strains:

• Amycolatopsis orientalisATCC 53884 (American Type Culture Collection)143

• Amycolatopsis mediterranei NRRL 18815 (Agricultural Research Service Patent Cul- ture Collection).143

Further constructs were subcloned from M3 ACP, M4 ACP and M5 using primers or- dered from IDT (Table 7.3). Primer suitability was checked using OligoAnalyzer 3.1 (IDT). Stocks of primers were diluted to 100µM (dH2O) and stored at−20◦C. Small aliquots of

primers were further diluted to 10µM before use (dH2O).

Table 7.3. Primers for all constructs used in this thesis. Bases shown inredcorrespond to stop codons and bases shown inblueallow the ligation of the PCR product into a TOPO vector.Site-directed mutationsare also highlighted.

Protein Plasmid name Origin gene Length (bp) Vector Forward primer Reverse primer

M3 ACP pTG5000 qmnA1 444 pET151 CACCTTG CTG GAC CTG GTC CTATCT TCT CCC GTT GCC M3 ACPT pTG5001 qmnA1 240 pET151 CACCTTG CTG GAC CTG GTC CTAGTC GGT GGG TTC CGG M4 ACP pTG5002 qmnA2 444 pET151 CACCCTG CTC ATC GAC GT CGG TGC TCA TGC GGT TT

M4 ACPT pTG5003 qmnA2 231 pET151 CACCCTG CTC ATC GAC GTG CTACGG GGC CAG TTC GG M5 ACP pTG5004 qmnA3 261 pET151 CACCACG GGT GCC GAG CA CTAGGA TGG TTC GGC GGC M5 KS pTG5005 qmnA3 1,377 pET151 CACCATG AGC ACC GGC CCG AC CTACCG TTC CAG TAT CAC GTG CGC GTT M5 KSATS pTG5006 qmnA3 2,664 pET151 TAA TAACGG GTC GAC CTG CCG ACG CCT GGC CCC GCT GAA CCC M5 KSAT pTG5007 qmnA3 3,594 pET151 TAAAAG GGC GAG CTC AGA TCC GG TTACGG CAC GCT CGC GCT CAG M5 pTG5008 qmnA3 3,876 pET151 CACCATG AGC ACC GGC CCG AC CTAGGA TGG TTC GGC GGC C207A pTG5008-11 qmnA3 3,876 pET151 CGT CAC CAT CGA CACCGCAGC CTC GTC CTC GCC GGT CCC TCC AGG CCG AAG GTG TAG G

S656C pTG5012-16 qmnA3 3,876 pET151 GCC GGT CAT TGCATC GGC GAG C ACA AAG GAC GGT CGA ACG C

M5 KSATS-A pTG5017 qmnA3 2,761 pET151 TAA TAACTC GCC GCC GAA CCA TCC CAG GTC GAC CTG CGC CGA

M5 KSATS-B pTG5018 qmnA3 3,202 pET151 TAA TAACTC GCC GCC GAA CCA TCC CAG GTC GAC CTG CGC CGA

M5 KSATS-C pTG5019 qmnA3 3,382 pET151 TAA TAACTC GCC GCC GAA CCA TCC GAG TTC CGC GCG CTC CAG

M5 KSATS-D pTG5020 qmnA3 3,418 pET151 TAA TAAGCC TGG GAC GGC GTC GCT GAC CAC CAC CGC ACC CCG

M5 KSATS-E pTG5021 qmnA3 3,480 pET151 TAA TAAGCT GCG GCA GGG CGC ACC AGG CCG ACC GCG CTC G

M5 KSATS-F pTG5022 qmnA3 3,531 pET151 TAA TAACTC GCC GCC GAA CCA TCC CAA CGG CGC CAA CGC CTC

M5 KRD_{-A pTG5023 qmnA3 717 pET151 CTG GCT GGT CCC GTC CGG GTG AAG GGA TCA ATT CCC TGA AAA TAC AGG TTT TC}

M5 KRD_{-B pTG5024 qmnA3 555 pET151 GTA CGC CTG CGA CCA CCT GTG AAG GGA TCA ATT CCC TGA AAA TAC AGG TTT TC}

M5 KRD_{-C pTG5025 qmnA3 408 pET151 CGA CGG CGA CTG GTG GCC GTG AAG GGA TCA ATT CCC TGA AAA TAC AGG TTT TC}

M5 KRD_{-D pTG5026 qmnA3 288 pET151 ACT GGC GGC GGT TGT CCA GTG AAG GGA TCA ATT CCC TGA AAA TAC AGG TTT TC}

A ChampionTMpET151 Directional TOPO®host-expression vector (pET151, Figure 7.1)191 was used for the overproduction of all proteins. Plasmids in pET151 undergo low-copy replication due to the presence of the basis of mobility (bom) region from pBR322 origin

(origin of replication) and repressor of primer (ROP) genes. The presence of aβ-lactamase gene (AmpR) ensures the production of an enzyme that inactivates β-lactam antibiotics, such as ampicillin, thus conferring an antibiotic resistance to E. coli cells containing the plasmid. This allows selection through lysogeny broth (LB) agar ampicillin plates of plasmid-containing cells, minimising the chance of selecting an empty vector.

Figure 7.1.A gene map of the pET151 D/TOPO vector (SnapGene).

must bind to allow transcription of the GOI to occur. This transcription is regulated by thelactose(lac) operon, which encodes a repressor of thelacoperator (lacI), preventing the binding of the T7 RNA polymerase to the T7 promoter site. As a result, overexpression of the gene cannot occur whilst the repressor is present, preventing leaky expression of the gene.

In addition, the presence of 6xHis encodes an in-frame His-tag at the protein’s N-terminus. A TEV site (ENLYFQG) is also encoded to allow the cleavage of a His-tag from the pu- rified recombinant protein, leaving an N-terminal tag of only 6 residues attached to the protein. A gene encoding a V5 epitope tag on the protein’s N-terminus is also present (V5 tag), allowing the tagged protein to be analysed by immunochemical methods, such as Western blot.

pJET1.2 also used to allow successful site-directed mutagenesis of longer constructs due to its significantly shorter backbone. The inclusion of restriction sites allowed the mu- tated inserts to be transferred back into the original pET151 vector.