4 MATERIALS AND METHODS
4.6 Cloning
In general, genes encoding proteins of interest were amplified from E. coli genomic DNA by PCR using corresponding primers containing cleavage sides for restriction enzymes at their 5' ends and the amplified DNA fragments were digested with the respective restriction enzymes (NEB). The target vector was cleaved with the respective enzyme, dephosphorylated by arctic phosphatase (NEB), and the DNA fragment was ligated into the vector using Quick T4 DNA ligase (NEB) according to the
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manufacturer’s protocol. The ligation mixture was transformed into E. coli TOP10 cells (Invitrogen) and plated on LB agar plates containing the antibiotic, for which the vector provides resistance.
Colonies were picked and grown over night in LB medium containing antibiotic and the plasmid was purified using the NucleoSpin Plasmid kit (Macherey-Nagel) according to the manufacturer’s protocol. The sequence of the plasmid was determined by DNA sequencing.
To introduce point mutations into plasmids by QickChange technique (Stratagene) (Constructs 08b-j) PCR primers which spanned the target position for mutation and contained the point mutation were generated. The mutation was introduced into the plasmid by PCR amplification of the complete vector using the primers. The template, not containing the mutation is digested by Dpn1, which digests methylated DNA, for 1-2 h at 37 °C. Plasmid multiplication and purification was performed as described above.
4.6.1 Construct for overexpression of EF-P and its modifying enzymes
The vector constructs containing genes encoding EF-P and its modifying enzymes EpmA and EpmB (pET28efp, pET28efp/epmA, pET28efp/epmA/B) were kindly provided by Frank Peske, MPI-BPC, Göttingen. The construct pET28efp/epmA/B/C was cloned on the basis of pET28efp/epmA/B by Christina Kothe, MPI-BPC Göttingen. The cloning strategy was as follows: Genes coding for EF-P (efp) and its modifying enzymes EpmA (epmA), EpmB (epmB) and EpmC (epmC) were amplified from the E.
coli genome (BL21(DE3) cells). The genes were first cloned separately into pET24a and a construct containing the ribosomal binding site encoded by pET24a together with the respective gene was cloned into pET28a. The efp gene was cloned into pET28a, adding an N-terminal 6xHis tag to EF-P as described (Yanagisawa et al, 2010). To obtain EF-P in different modification statuses, EF-P was overexpressed either alone or together with its modifying enzymes from the following constructs:
pET28efp, pET28efp/epmA, pET28efp/epmA/B and pET28efp/epmA/B/C (Fig. M1). Since only EF-P need to be purified, the other genes were cloned without tag.
Fig. M1 EF-P constructs in pET28a
4.6.2 Generation of mRNA constructs
To generate mRNAs corresponding to proteins containing polyproline motifs the respective genes (encoding for release factor glutamine methyltransferase (PrmC; 1-75); TonB, outer membrane subunit Rz1; N-acetylmuramoyl-L-alanine amidase (AmiB, 1-159); flagellar transcriptional regulator (FlhC, 1-94); flagellar regulator (Flk, 1-87) and YafD (1-75)) were amplified from genomic DNA of E.
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coli BL21(DE3) with primers introducing 3' Xho1 and 5' Nde1 restriction sites (Table M5). The PCR product was digested with Nde1 and Xho1 (New England Biolabs) and ligated into the pET24a vector (Novagen). The gene of secreted effector protein EspF(U) (1-154) from E. coli DH5alpha with 3' Xho1 and 5' Nde1 restriction sites was purchased from Eurofins and cloned into pET24a as described for the other constructs. Some of these constructs were cloned by Christina Kothe and Jörg Mittelstät, MPI-BPC Göttingen (see Table M4).
4.6.3 Construction of tRNA
Protemplate
The transcript of tRNAPro was prepared as described (Brown et al, 1979). Two partially complementary DNA oligomers containing isoacceptor tRNAPro with the anticodon CGG fused to T7 RNA polymerase promoter were obtained from IBA (primer 1 and 2,Table M5), extended by PCR and cloned into pUC19 vector using SMA1 restriction site (Himeno et al, 1989). The CCG isoacceptor was chosen because its matching codon (CCG) is the most abundant of the four Pro codons (Dong et al, 1996) and was reported to be the most efficient for proline incorporation (Pavlov et al, 2009).
4.6.4 Constructs used in this study
Table M4: Plasmids
C Construct Comment
01 pET28_efpa pET28 vector for expression of N-terminally 6xHis-tagged EF-P 02 pET28_efp/epmA/Ba Like 01 but additionally for expression of EpmA and B
03 pET28_efp/epmA/B/Cb Like 01 but additionally for expression of EpmA, B and C 04 pET24a_epmAa pET24a vector containing EpmA gene, template for cloning
EpmA with ribosomal binding site (RBS) of pET24a into pET28 05 pET24a_epmBa Like 04 but encoding EpmB instead of EpmA
06 pET24a_epmCb Like 04 but encoding EpmC instead of EpmA
07 pUC19_tRNAPro pUC19 vector containing sequence of tRNAPro CGG isoacceptor fused to T7 RNA promoter
08a pET24a_prmCc pET24a vector containing sequence of PrmC, template for mRNA formation
08 b-j pET24a_prmC_mod Like 08a, containing pointmutations introducing PG, PP, PPG, PPGF and PPP into PrmC sequence
09-15 pET24a_XYb pET24a vector containing sequence of genes coding for TonB (09), YafD (10), Rz1 (11), AmiB (12), FlhC (13), Flk (14) and EspfU (15)
16 pEX-K_EspfU1-154 pEX-K vector containing sequence of EspfU gene from E. coli DH5alpha (N-terminal fragment, 154 amino acids)
Constructs were a provided by F. Peske; cloned by b C. Kothe or c J. Mittelstät (Mittelstaet, 2012)
4.6.5 Primer
DNA oligonucleotides were obtained from IBA Life Sciences or Eurofins MWG Operon.
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Table M5: DNA oligonucleotides
Primer for cloning EF-P constructs
Name Sequence 5'→ 3' Template/Comment
EF-P_Nde1_F ATTCCTCATATGGCAACGTACTATAGCAA CGATTTTCG
For amplification from E. coli BL21(DE3) genome and cloning into pET28 by N-terminal Nde1 and C-N-terminal Yba1
For amplification from E. coli BL21(DE3) genome and cloning into pET24a by N-terminal Nde1 and C-N-terminal Yba1
To remove internal Nde1 site in EpmA gene
F:EpmA including RBS, R: introduces Stop codon and EcoR1 site
EpmA_pET28_R TGGTGGGAATTCTCTTATGCCCGGTCAAC GCTAAAGGC
EpmB_pET24a_F GAGATATACATATGGCGCATATTGTAACC CTAAATACCCC
For amplification from E. coli BL21(DE3) genome and cloning into pET24a by N-terminal Nde1 and C-N-terminal Yba1 RBS, introduces EcoR1 site; R:
introduces Stop and BamH1 site EpmB_pET28_R GGTGGTGGATCCCTTACTGCTGGCGTAG
C
EpmC_pET28_F AGTTGCTAGCAAGGAGATATACATATGA ACAGTACACACCACTACGAGCAGTTG
For amplification from E. coli BL21(DE3) genome and cloning into plasmid 02 by N-terminal Nde1 and C-terminal Pst1 restriction sites
EpmC_pET28_R TCAACTGCAGTCAGTTGAGCGCTTCCGGC C
Primer for tRNA transcript
Name Sequence 5'→ 3' Template/Comment
1 AGTTGCTGCAGTAATACGACTCACTATAC
GGUGAUUGGCGCAGCCUGGUAGCGCAC UUCGUUCG
For cloning of tRNAPro sequence into PUC19 using SMA1 restriction sites
2 TGGTCGGTGATAGAGGATTCGAACCTCC
GACCCCTTCGTCCCGAACGAAGTGCGCTA CCAGGCTG
3 GTTTTCCCAGTCACGAC pUC19_tRNAPro, to generate PCR
template for transcription
4 TGmGTCGGTGATAGAGGATTC
m designates a 2'-O-methyl group to prevent nonspecific 3'mRNA extension by T7 RNA polymerase Primer for cloning translation constructs into pET24a
Name Sequence 5'→ 3'
tonB_Nde1_F GATTATGACTCATATGACCCTTGATTTACCTCGCCG
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Quickchange primers to modify 08a pET24a_prmC construct
Name Sequence 5'→ 3'
T7-5 forward TAATACGACTCACTATAG Forward primer for all mRNAs PrmC 14_R AAGTTGGCTTATTGCTTCACG pET24a_PrmC, generates template
for mRNA of indicated length
69 PrmC 224_R CGACTGTTCGATGATATGCACGA PrmC 252_R GAGGATAAATGCTTGTCGCACCG
TonB 56_R TTCGAGATCAGCAGGCGTAACC pET24a_TonB, generates template for mRNA of indicated length
YafD_20_R TAAGATCCTTTCCGCAGGTTGTC pET24a_YafD, generates template for mRNA of indicated length
yafD_75_R CAATAACACCAGATGTGCATC
Rz1_40_R GCATTATCCACGCCGGAGG pET24a_Rz1, generates template
for mRNA of indicated length Rz1_62_R GTTCCCGGAGGGTGAAATAATCC
AmiB_90_R AGGCGTTCCAGAGCGAATC pET24a_AmiB, generates template
for mRNA of indicated length AmiB_129_R CACATCGGCGTTAATCGTAAAGA
AmiB_159_R GCGCGGTGCGACAACC
FlhC_52_R CGGTGGGCTTCCGCGCAGTTC pET24a_FlhC, generates template for mRNA of indicated length
FlhC_94_R CGCATCGACGCCATTACACA
Flk_14_R TGGTGGTTGCCCAGGAGG pET24a_Flk, generates template for mRNA of indicated length
Flk_94_R ACGAACCAGCCAGACCAG
EspfU_117_R TGGTGGCGCAGGCCAGTTAG pET24a_EspfU, generates template for mRNA of indicated length EspfU_155_R CTCGAGGAATATGTTCAGCCATAT