2. Materials and Methods
2.4 Cloning strategies and construction of plasmids
Positive transformants were selected via colony PCR and the correct sequence of the extracted plasmid DNA was confirmed by sequencing.
2.4.3 General strategy for construction of knockout mutants of small ncRNAs
In order to create knockout strains of Hfq-‐dependent putative sRNAs (Hprs) in Synechocystis upstream and downstream genome regions were amplified using Synechocystis genomic DNA and then fused together via a third PCR, which resulted in omission of the sRNA. The following primer combinations were used:
Hpr8: 1.PCR slr1213-‐fw slr1213-‐salI-‐rev 2.PCR slr1214-‐salI-‐fw
slr1214-‐rev 3.PCR slr1213-‐fw
slr1214-‐rev
Hpr10: 1.PCR sll1834-‐fw sll1834-‐BglII-‐rev 2.PCR slr1915-‐BglII-‐fw
slr1915-‐rev 3.PCR sll1834-‐fw
slr1915-‐rev (Table 4)
PCR products were ligated into the pJet 1.2 (in case of Hpr8) and pDrive (for Hpr10) cloning vectors. Recombinant vector was transferred into competent E.
coli DH5α, positive transformants harbouring the respective recombinant vector were selected on LB agar medium supplemented with Amp and checked via colony PCR. Plasmid vector DNA was then extracted and digested with SalI and BglII restriction enzymes (both Thermo Fisher Scientific) respectively. The Km resistance gene cassette was obtained from the pUC4K vector, restricted with SalI or BamHI, respectively. The Km resistance gene cassette was then ligated into the digested pJet vector. Competent E. coli DH5α were transformed with recombinant vector, positive transformants were selected on LB agar medium supplemented with Amp and Km and checked via colony PCR. Then the plasmid vector was extracted, sequenced and used to transform Synechocystis. Positive transformants were selected on BG11 agar medium supplemented with Km and checked via colony PCR.
2.4.4 General strategy for construction of overexpression mutants of small ncRNAs
In order to create overexpression strains of sRNAs in Synechocystis their ORFs were amplified using Synechocystis genomic DNA and primer combinations leading to the introduction of copper-‐regulated petJ promoter in front of the sRNA followed by the oop-‐terminator from phage Lambda:
Hpr8: 1.PCR PpetJ-‐fw
Hpr8-‐PpetJ-‐rev 2.PCR PpetJ-‐Hpr8-‐fw
Oop-‐Hpr8-‐rev 3.PCR PpetJ-‐fw
Oop-‐Hpr8-‐rev Hpr10: 1.PCR PpetJ-‐fw
Hpf10-‐PpetJ-‐rev 2.PCR PpetJ-‐Hpr10-‐fw Oop-‐Hpr10-‐rev 3.PCR PpetJ-‐fw
Oop-‐Hpr10-‐rev (Table 4)
Final PCR products were ligated into the pDrive cloning vector. Recombinant vector was transformed into competent E. coli DH5α cells, positive transformants harbouring recombinant vector were selected on LB agar medium supplemented with Amp and checked via colony PCR. Plasmid vector was then extracted and digested with PstI and SalI restriction enzymes (both Thermo Fisher Scientific) and inserted into the conjugative, self-‐replicating vector pVZ321-‐Strep digested with the same restriction enzymes. It was then transformed into competent E. coli DH5α cells, positive transformants were selected on LB agar medium supplemented with Km and Strep and checked via colony PCR and sequencing. Then the extracted plasmid vector was transferred into Synechocystis WT and Δhfq mutant by conjugation. Positive conjugants were selected on BG11 agar medium supplemented with Km and Strep and checked via colony PCR.
2.4.5 Construction of slr1214-‐rescue and hpr8-‐rescue mutants in Synechocystis
Hpr8-‐rescue and slr1214-‐rescue strains on the basis of the Δhpr8 mutant were constructed by Jasper Matthiessen (AG Hess, Institute of Biology III, Albert-‐
Ludwigs University Freiburg) by introducing the self-‐replicating plasmid pVZ322, modified to carry the native hpr8 locus containing native hpr8 promoter in front of hpr8 (PHpr8>Hpr8). hpr8 locus was amplified by PCR using the primers P-‐Hpr8-‐MluI-‐fw and SyR14-‐MluI-‐rev (Table 4) that introduce MluI restriction sites on both ends of the PCR product. The PCR product and pVZ322 were then digested with MluI, thus removing the Km resistance gene cassette from pVZ322. Following that, the fragments were ligated and transformed into E.
coli TOP10F’. Positive clones were selected with Gent and verified by colony PCR and sequencing. Recombinant plasmid was then transferred via conjugation into Δhpr8 and positive clones were selected using Km and Gent.
In order to create slr1214-‐rescue strain hpr8 promoter was fused directly to the coding sequence of slr1214 (PHpr8>slr1214), omitting the hpr8 sequence in between the two. This was executed by introducing the self-‐replicating plasmid pVZ322 carrying the PHpr8>slr1214 construct into the Δhpr8 knockout mutant.
The promoter of hpr8 was amplified using the primers PHpr8-‐MluI-‐fw and PHpr8-‐Fus-‐rev (Table 4), introducing a MluI restriction site on the 5’-‐end and a
complementary region for fusion-‐PCR on the 3’-‐end of the PCR product. The coding region of slr1214 was amplified using the primers slr1214-‐Fus-‐fw and slr1214-‐MluI-‐rev, thus introducing MluI restriction site on the 3’-‐end and a complementary region for fusion-‐PCR on the 5’-‐end of the PCR product. The two products were fused together by PCR. This final product and pVZ322 vector were then digested with MluI, ligated and transformed into E. coli TOP10F’. Positive clones were selected with gentamycin and verified by colony PCR and sequencing. Finally the correct, recombinant plasmid was conjugated into the Δhpr8 knockout mutant and positive clones selected using kanamycin and gentamycin.
2.4.6 General strategy for construction of RNaseIII conditional knockout mutants in Synechocystis
First step in creation of conditional knockout strain of RNaseIII in Synechocystis was to construct a complementation mutant of RNaseIII in the existing knockout mutant. For that purpose FLAG-‐slr0346 plasmid was transferred to Synechocystis Δslr0346(Cmr) via conjugation and FLAG-‐slr1646 plasmid was transferred to Synechocystis Δslr1646(Cmr) also via conjugation. Both mutants were grown on BG11 medium without copper, thus inducing the expression of the respective RNasesIII controlled by the petJ promoter. In the next step knockout strains of RNasesIII (slr0346 and slr1646) were created using the following strategy:
upstream and downstream genome regions were amplified using Synechocystis genomic DNA and then fused together via a third PCR, thereby skipping RNaseIII.
The following primer combinations were used:
rnc1 (slr0346): 1.PCR slr0346-‐k/o-‐fw slr0346-‐rev-‐StuI
2.PCR slr0346-‐fw-‐StuI slr0346-‐k/o-‐rev 3.PCR slr0346-‐k/o-‐fw
slr0346-‐k/o-‐fw rnc2 (slr1646): 1.PCR slr1646-‐k/o-‐fw slr1646-‐StuI-‐rev 2.PCR slr1646-‐StuI-‐fw
slr1646-‐k/o-‐rev 3.PCR slr1646-‐k/o-‐fw
slr1646-‐k/o-‐rev (Table 4)
PCR products were ligated into the pJet 1.2 cloning vector. Recombinant vector was transformed into competent E. coli DH5α, positive transformants harbouring recombinant vector were selected on LB agar medium supplemented with Amp and checked via colony PCR and sequencing. Plasmid vector was then extracted and digested with StuI restriction enzyme (Thermo Fisher Scientific). Gent resistance cassette was obtained from the pVZ322 vector, restricted with StuI and HincII restriction enzymes (both Thermo Fisher Scientific). Gent resistance cassette was then ligated into the digested pJet. Recombinant vector was transformed into competent E. coli DH5α, positive transformants were selected
on LB agar medium supplemented with Amp and Gent and checked via colony PCR and sequencing.
In the final step Δslr1646(Gentr) plasmid was transformed in Synechocystis FLAG-‐
slr0346 + Δslr0346(Cmr), as well as Δslr0346(Gentr) plasmid was transformed in Synechocystis FLAG-‐slr1646 + Δslr1646(Cmr). Positive transformants were selected on BG11 agar medium supplemented with Gent, Cm, Km and lacking copper. Thus when these mutants were transferred to the growth medium with normal copper concentration the expression of RNaseIII under the control of petJ promoter was abolished and conditional knockout of both RNasesIII was achieved.