Curvas de calibración Si bien la linealidad es un parámetro importante, las curvas de calibración son determinantes a la hora de estandarizar y

Five plasmids were constructed in this work to produce an a-ketoglutarate dehydrogenase over-expression vector, and a red pigment knock-out vector. The various components (e.g. genes, sites) assembled in the final plasmids are listed and described Table 42.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

Table 42: Plasmid component definitions.

Plasm id Com ponent Description

flc/II-0RF4 Gene that encodes protein that activates Pact/and V a c t i i p

P a c t i Site that protein, resulting from acdI-0R F4 gene, binds to for initiating replication of gene o f interest.

P a c t l l l Second site that transcribes in opposite direction of Pacf/on dsD N A .

oriC Origin o f replication for E. coli. Includes all genes and sites

necessary for a plasmid to replicate in E. coli.

oriT Origin o f transfer from E. coli to another bacteria cell.

Needed for conjugation. All other required machinery (except oriT) for conjugation are on pUZ8002 in E. coli strain ET12567. This is to reduce the number of genes needed on plasmid of interest but still allow conjugation. pUZ8002 will not be transferred from E. coli.

int (VWB) Gene that codes for integrase protein which allows specific integration of plasmid DNA into genomic DNA. Integration is important to ensure that DNA of interest is replicated as single copy.

attP (VWB) Site on plasmid that integrase binds to for integration. There is an attB VWB site in S. erythraea genome that the same protein binds to for specific site directed integration. P = plasmid and B = bacteria.

aprR Apramycin resistance gene.

tsrR Thiostrepton resistance gene.

ampR {bid) Ampicillin resistance gene.

rppA fragment of RppA red pigment synthesis enzyme.

a-ketoglutarate dehydrogenase gene {S. coelicolor)

E l and E2 subunits of a-ketoglutarate dehydrogenase gene from S. coelicolor.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

6.4.1 a-Ketoglutarate Dehydrogenase Over-expression in S. erythraea

6.4.1.1 pMU1

p M U l, shown in Figure 53, is an integrative vector containing VWBint, attB VWB,

o h C , oriT, and aprR. pM U l was constructed by digesting pMG55 with Nhe I and

Apa I and pKT02 with Xba I and Apa I. The 3.8 kb fragment o f pMG55, containing

oriC, oriT, and aprR, was SAP treated and ligated to the 2.2kb fragment o f pKT02, containing VW Bint and attP VWB. pM U l is apramycin resistant in both E. coli and

S. erythraea. o r i T cittP(VWB) int (VWB) ,4cr 651(681) 718(681) Kpn 1 (685) Hi,I d 111 (687) Xha 1 (709) \ (Vo/ 1 (722) Eco RV(735) /tc6 1 (739) A p o 1 (739) Eco R I (739) o r i C

Figure 53: Diagram of pM Ul.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

6.4.1.2 pMU2

pM U 2 is an a-ketoglutarate dehydrogenase coding sequence plasm id containing the P C R gene product o f the S. coelicolor a-ketoglutarate dehydrogenase w hich has a m ethionine start codon engineered at the beginning o f the sequence using an N de I site, and dtnXba I site at the end for excision from the interm ediate vector pUC18. The S. coelicolor a-ketog lu tarate dehydrogenase sequence w as am plified b y PC R using S. coelicolor genom ic D N A as the tem plate and oligonucleotides M U SCLF and M U SC L R as prim ers w hich introduce the N de I and X ba I restriction sites. The 3.8 kb P C R fragm ent w as isolated and 5 ’-phosphorylated w ith T4 polynucleotide kinase. It w as then ligated to Sm a I digested and SAP treated pU C 18 and confirm ed b y sequencing. pM U 2 is am picillin resistant in E. coli and contains la c Z a for blue-w hite selection in the presence o f IPTG and X-gal.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

6.4.1.3 pMU3

pMU3, shown in Figure 54, is an expression and integrative vector containing the com ponents o f pM U l plus the promoter system <2c/II-ORF4, P a c t i , and ? a c t i i - pMU3

was constructed by digesting pM U l with Sph I and Xba I and pCJW 90 with Sph I and

Spe I. The 5.4 kb fragment from pM U l was then ligated to the 1.5 kb fragment from pCJW90. pMU3 is apramycin resistant in E. coli and S. erythraea. pMU3 contains all the necessary components to over-express a gene using the Vacti promoter system and to specifically integrate the plasmid DNA using the VWB integrase system (so long as the bacterial chromosome contains an attB binding site).

o riC Xba 1(540 0) ac /II-0R F 4 a c t l l l Nde 1 (5641 ) a ttP (VW B) aprR int (VW B)

Figure 54: Diagram of pMU3.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

6.4.1.4 pMU5

pM U5, shown in Figure 55, is an S. coelicolor a-ketoglutarate dehydrogenase expression and integrative vector containing the components o f pMU3 plus the S. coelicolor a-ketoglutarate dehydrogenase gene sequence over-expressed using the

^acti promoter system. pMU5 was constructed by digesting both pM U2 and pMU3 with Nde I and Xba I. The 6.8 kb fragment from pMU3 was SAP treated and ligated to the 3.8 kb fragment o f pMU2. pMU5 is apramycin resistant in E. coli and S. erythraea. 1(1) a-ketoglutarate DH [S. c o e l i c o l o r ) N de 1 (6646) P actI P actlll flc/II-0R F4 p M U 5

10466 bp

Figure 55: Diagram of pMU5.

Ph.D. Thesis M etabolic Engineering o f S. erythraea

6.4.2 Red Pigment Synthase Gene Knock-Out In S. erythraea

6.4.2.1 pMU4

pM U4, shown in Figure 56, is a recom bination vector containing oriC, oriT, ampR, tsrR, and 1 kb o f sequence hom ology to rppA o f S. erythraea. The 1 kb rppA

fragment was amplified by PCR using S. erythraea genomic DNA as the template, and M USERFF and M USERPR as the prim ers which also engineered AZir/1 restriction sites at each end. pM U4 was constructed by digesting pCJW 192 with I and Spe I, and digesting the isolated PCR fragment with X ba I. Xba I and Spe I have compatible sticky ends that can be ligated but then do not reform either site. The 4.1 kb pCJW 192 fragment was SAP treated and ligated to the 1 kb PCR fragment. pMU4 is ampicillin resistant in E. coli and thiostrepton resistant in S. erythraea.

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