Lars Röse: Role of Undecaprenyl Phosphokinase in mycobacteria: impact on biofilm formation, growth properties, persistence, and virulence
[Front page] Chapter: 1. | 2. | 3. | 4. | 5. [Table of abbreviations] [References] [Danksagung] [Selbständigkeitserklärung] [Appendix]
Page: [Table of contents]
[page 116↓]

Appendix

Tables of strains, plasmids, and primers:

Table 4: Strains and plasmids used in this study

Strain or Plasmid

Descriptiona

Source or Reference
   

Strains

  

E. coli DH5α

standard cloning strain.

Invitrogen

E. coli HB101

cloning strain for resolvase instable plasmids

 

M. smegmatis mc2155

wildtype, ept-1

[40]

M. smegmatis mc2155 pYUB657-Δupk

single crossover strain, HygR, SucS

This study

M. smegmatis mc2155 Δupk

deletion mutant of the upk gene, HygS, SucR

This study

M. smegmatis mc2155 Δupk + pMV262-rv2136c

reconstitution of the upk deletion mutant, HygS, SucR, KanR

This study

M. tuberculosis H37Rv

wildtype, standard laboratory strain

 

M. tuberculosis H37Rv Δupk

deletion mutant of upk gene, HygR

This study

M. tuberculosis H37Rv Δupk + pMV262-rv2136c

reconstitution of the upk deletion mutant, HygR, KanR

This study

M. bovis BCG

standard vaccine strain

 

M. bovis BCG Δupk

deletion mutant of upk gene, HygR

This study
   

Plasmids

  

pPCR-Script Amp

Cloning vector, AmpR

Stratagene

pYUB657

suicide plasmid, bearing groEL2 (Hsp60) promoter and sacB , HygR, AmpR

[58]

pYUB657-Δupk

suicide plasmid carrying upk-deletion template

This study

pMV262-rv2136c

pMV262-Kan containing the M. tuberculosis rv2136c gene

This study

pMV262-Kan

E. coli-mycobacteria shuttle vector, groEL2 (hsp60) promoter, KanR, ColE1, OriM

W.R. Jacobs Jr.

pJSC284

starting plasmid for construction of a knockout template, HygR

W.R. Jacobs Jr.

pKO-upk

pJSC284, containing flanking regions, HygR

This study

a AmpR, ampicillin resistance; KanR, kanamycin resistance; HygR, hygromycin resistance

[page 117↓]

Table 5: List of primers

    

Amplified DNA fragment

Primera

Template DNA

    

M. smegmatis

   

flanking region 1

P1: 5’-GATATCcttttcgagcacgaccga-3’

P2: 5’- AAGCTTcttggcaccggggtactg -3’

M. smegmatis mc 2155

flanking region 2

P3: 5’-AAGCTTcatcgcaggaacgtcagt-3’

P4: 5’-GATATCaggtcgacacggaagtagcca-3’

M. smegmatis mc 2155
   

M. tuberculosis

  

flanking region 1

P1: 5’-GGATCCcgtcatcgaagaccatga-3’

P2: 5’-GGATCCgacttgccaccaagacat-3’

M. tuberculosis H37Rv

flanking region 2

P3: 5’-ACCGGTctcgtgctgctggctacc-3’

P4: 5’-ACCGGTcacctccaaaataccgcg-3’

M. tuberculosis H37Rv

rv2136c

rv2136c-START: 5’-GGATCCatgctgtggcacgcaatg-3’

rv2136c-END: 5’-GGATCCccagtcgtcctcttcgtc-3’

M. tuberculosis H37Rv
   

RealTime

  

rv2136c

rv2136c-fw: 5’-atcctgagcgcttggttg-3’

rv2136c-rev: 5’-gggtttgggcaataccaac-3’

M. tuberculosis H37Rv wildtype, Δupk mutant, reconstitution

rv3627c

rv3627c-fw: 5’-ccacagtcaaggcgggagtggt-3’

rv3527c-rev: 5’-acacgacaggtccctgggggtt-3’

M. tuberculosis H37Rv wildtype, Δupk mutant, reconstitution

rv2946c

rv2946c-fw: 5’-aactcgagtctgccggtg-3’

rv2946c-rev: 5’-tagcaccgaggcatccac-3’

M. tuberculosis H37Rv wildtype, Δupk mutant, reconstitution

a capital letters indicate the added restriction sites

[Front page] [1.] [2.] [3.] [4.] [5.] [Table of abbreviations] [References] [Danksagung] [Selbständigkeitserklärung] [Appendix] [Table of contents]

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