Construction of Plasmids

Table 8.3: Details of the construction of plasmids made during this thesis.

Plasmids that are listed in Table 8.1 and made during this thesis are aligned with the method used, primers (listed in Table 8.2), the template and the purpose of the mutagenesis. The corresponding sequencing primers (Seq. Primer) can also be found in the list. Methods: QC = Quik Change (Ch. 3.2.3.10); Inv = Inverse PCR (Ch. 3.2.3.11); Sub = Subcloning (Ch. 3.2.3.1 to 3.2.3.6).

Product Method Primer Template Purpose Seq.

Primer

region (in front of internal His6 -tag)

C3/C6

pCLA12 QC C13 + C14 pCLA9 deletion of 20 AAs of the linker region (in front of internal His6 -tag) pCLA33 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA34 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA35 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53

pCLA36 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA37 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA38 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA39 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA40 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA41 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA42 Inv C48 + C51 pCLA72 MjYRS_AGGA promoter library C52/C53 pCLA44 Inv C54 + C55 pCLA79 PylT promoter library C57 pCLA53 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA54 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA55 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA56 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA57 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA58 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA59 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA60 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA61 Inv C56 + C55 pCLA82 MjYT_UCCU promoter library C57 pCLA62 Inv C58 + C59 pCLA85 glnS promoter in front of pylS pCLA72 QC C75 + C76 pCLA71 SalI restriction site behind

MjYRS terminator

C3/C53 pCLA73 QC C77 + C78 pCLA9 NdeI restriction site in front of

PylS start codon changed to KpnI

C3/C6 pCLA74 QC C79 + C80 pCLA73 PstI restriction site behind PylS

stop codon changed to SacI

C50/C53

pCLA75 QC C81 + C82 pCLA74 SalI restriction site in front of PylS promoter

C6/C50 pCLA76 QC C83 + C84 pCLA75 NotI restriction site behind PylS

terminator pCLA79 QC C90 + C91 pCLA78 MfeI restriction site behind PylT

terminator pCLA82 QC C94 + C95 pCLA81 XhoI restriction site behind

MjYT_UCCU terminator

C57/C89 pCLA83 QC C81 + C82 pCLA9 SalI restriction site in front of

PylS promoter

C6/C50 pCLA84 QC C83 + C84 pCLA9 NotI restriction site behind PylS

terminator pCLA87 QC C100 + C101 pCLA76 point mutation in restored NdeI

site

C50/C53 pCLA88 QC C102 + C103 pCLA76 point mutation in restored NdeI

site

C50/C53 pCLA89 QC C104 + C105 pCLA76 point mutation in restored NdeI

site

C50/C53 pCLA90 QC C106 + C107 pCLA76 point mutation in restored NdeI

site

C50/C53

pCLA92 Sub - pCLA72 MjYRS_AGGA gene cloned with

BamHI + SalI into pCLA91 pCLA106 Sub C119 + C120 pCLA102 sfGFP 150TAG gene cloned with

NcoI + HindIII into pCLA100

C117/C118 pCLA107 Sub C119 + C120 pCLA103 sfGFP 134AGGA 150TAG gene

cloned with NcoI + HindIII into pCLA100

C117/C118

pCLA108 Sub C119 + C120 pCLA104 sfGFP 134AGGA gene cloned with NcoI + HindIII into pCLA100

C117/C118 pCLA112 QC C121 + C122 pCLA111 Codon for T6 in the CAT gene

exchanged for TAG

C125-C127

pCLA113 QC C123 + C124 pCLA112 Codon for D111 in the CAT gene exchanged for AGGA

C125-C127 pCLA115 Sub - pCLA24 pylS with PglnS exchanged for

pylS with Plib3E by cloning into pCLA95 with SalI + NotI (+ XhoI)

pCLA120 Sub C128 + C129 pCLA119 MjAzFRS gene cloned with NdeI + PstI into pCLA1

C3/C6 pCLA121 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA122 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA123 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA124 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA125 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA126 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA127 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA128 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA129 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA130 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA131 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA132 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA133 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA134 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA135 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA136 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA137 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA138 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA139 Inv C130 and

C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6

pCLA140 Inv C130 and C131 + C132

pCLA120 MjAzFRS library in two rounds of inverse PCR

C6 pCLA142 QC C133 + C134 pCLA141 Codon for T6 in the H3opt gene

exchanged for AGGA

C108 pCLA143 Sub C135 + C136 pCLA142 H3opt T6AGGA gene cloned with

NcoI + HindIII into pCLA100

C117 pCLA144 Sub - pCLA122 MjYRS gene on pCLA98 replaced

by MjAzFRS gene (clone #2) with NdeI + StuI

digest only

pCLA145 Sub - pCLA123 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #3) with NdeI + StuI

digest only

pCLA146 Sub - pCLA125 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #5) with NdeI + StuI

digest only

pCLA147 Sub - pCLA126 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #6) with NdeI + StuI

digest only

pCLA148 Sub - pCLA131 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #11) with NdeI + StuI

digest only

pCLA149 Sub - pCLA137 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #17) with NdeI + StuI

digest only

pCLA150 Sub - pCLA138 MjYRS gene on pCLA98 replaced by MjAzFRS gene (clone #18) pCLA165 Sub C81 + C84 pCLA158 BCNRS gene cloned into pCLA154

with SalI + NotI

pCLA172 Sub - pCLA168 BCNRS gene with Plac cloned into pCLA154 with SalI + NotI (+ XhoI)

C111-C114 pCLA173 Sub - pCLA169 AzpcKRS gene with Pcon cloned

into pCLA154 with SalI + NotI (+ XhoI)

C111-C114

pCLA174 Sub - pCLA170 AzpcKRS gene with Plac cloned into pCLA154 with SalI + NotI (+ XhoI)

C111-C114

pCLA184 Sub C137 + C138 pCLA182 SmR gene on pCLA116 replaced by CatR gene with AgeI + NheI

C111/

C141/C142 pCLA185 Sub C139 + C140 pCLA183 SmR gene on pCLA116 replaced

by AmpR gene with AgeI + NheI

C111/

C141/C142 pCLA186 Sub C137 + C138 pCLA182 SmR gene on pCLA171 replaced

by CatR gene with AgeI + NheI

C111/

C141/C142 pCLA187 Sub C139 + C140 pCLA183 SmR gene on pCLA171 replaced

by AmpR gene with AgeI + NheI

C111/

C141/C142 pCLA193 QC C143 + C144 pCLA101 Embedding ORBS while “killing”

the WT Shine-Dalgarno sequence C145 pCLA194 QC C146 + C147 pCLA193 sfGFP gene exchanged by

sequence for GCN4 16-31

C145

Im Dokument A method for the genetically encoded incorporation of FRET pairs into proteins (Seite 172-178)