Manlio Fusciello Jaakko Itkonen Maarja Laos Tania Quirin Cloning CLUB Manlio Fusciello Jaakko Itkonen Maarja Laos Tania Quirin Group 3 11th October 2016

Aim To create 2 expression plasmids for expression of: 1) Fusion protein between Heat-shock protein 27 (Hsp27) and C-terminal intein NpuNΔ16, 2) Fusion protein between Heat-shock protein A1 (HspA1) and C-terminal NpuNΔ16 using pIVEX_GAA_Omega_eYFP-His as a backbone Created plasmids will be used for cell-free protein synthesis in tobacco BY-2 cell lysates

Strategy 1: Standard Nebuilder 2 Fragment Assembly Vector pIVEX_GAA_Omega_eYFP_His linearized and amplified with PCR, resulting in the removal of the eYFP_His insert Hsp27 and HspA1 PCR-amplified with primers that introduce 5’-overhangs with complementarity to the 3’ end of the plasmid, and to the 5’ end of the NpuNΔ16 Intein NpuNΔ16 PCR-amplified with primers that introduce 5’-overhangs with complementarity to the 3’ end of the HspXX and to the and 5’ end of the plasmid

The Destination

The PCR Generate DNA fragments with appropriate 5’-overhangs by PCR

The Assembly Gibson Assembly Master Mix 5’ exonuclease DNA polymerase DNA ligase

The Product? Assemblies were attemped twice e.g. with varying amounts of inserts and vector, and different reaction time Attempt yielded no colonies! Strategy 2!

Strategy 2: Overlap Extension PCR Instead of using 2 fragments in the Gibson Assembly, the fragments can be fused together in a separate PCR-reaction, and then used as a single fragment in the assembly reaction Template generation: Fragments are PCR-amplified with primers that introduce 5’- overhangs complementary to the fusion partners

Strategy 2: Overlap Extension PCR 25-35 PCR Cycles

Strategy 2: Overlap Extension PCR 1nd round of OE-PCR: First few cycles of PCR are run only with the template DNA fragments which, due to the generated 5’- overhangs, are able to prime each other 98° C Annealing

Strategy 2: Overlap Extension PCR The fragments get ’sown’ together DNA Polym. DNA Polym. 5-10 PCR rounds 72° C

Generated single fragment is used e.g. in a routine Gibson Assembly 2nd round of PCR: Either the generated DNA from the previous PCR is used as a template, or the reaction is merely topped up with the ’end’-primers, resulting in the amplification of the full fusion construct Generated single fragment is used e.g. in a routine Gibson Assembly 98° C DNA Polym. Annealing 72° C DNA Polym. ≈25 PCR Cycles later… µg of DNA

Strategy 2: Results Strategy 3! With both Hsps, faint hazy bands corresponding to the expected sizes of the HspXX_NpuNΔ16 fusions were observed on the agarose gel (HspA1_NpuNΔ16 shown) After excision and gel prep, the concentrations were deemed too low to be used for Gibson Assembly Further progress using this strategy was halted Possible ways to optimize the reaction include increased reaction volumes, Ta optimization, adjusting the 5’-overhang length etc. 3 kb 2 kb 1.5 kb 1 kb ~2.4 kb Strategy 3!

Strategy 3: Re-thought Nebuilder Plasmids containing other GOIs fused with the NpuNΔ16 were generated earlier during the project pIVEX_GAA_Omega_CNTF_NpuNΔ16 was chosen to be used as a plasmid backbone For HspA1, restriction sites NcoI and BbvCI can be applied for cut & paste cloning For Hsp27, no convenient restriction sites available, and we didn’t want to create a de novo site upstream. Thus, the chosen approach was to remove CNTF with Inverse PCR and to fuse the PCR-amplified Hsp27 with Gibson Assembly Since this yielded colonies, the same approach was applied to HspA1

Strategy 3: Re-thought Nebuilder

Strategy 3: Assembly

Strategy 3: Results Hsp27_NpuNΔ16 HspA1_NpuNΔ16 Following transformation, plenty of colonies were observed on LB-Amp plates Colony PCR was carried out to verify insertion of fragments Expected band sizes of the amplified inserts: Hsp27_NpuNΔ16 ~1100 bp HspA1_NpuNΔ16 ~2400 bp 3 arbitrarily chosen colonies from each plate were grown, prepped and sent for sequencing → Sequencing verified succesful assembly! Hsp27_NpuNΔ16 HspA1_NpuNΔ16 3 kb 3 kb 2.5 kb 2 kb 2 kb 1.2 kb 1 kb 1 kb

Strategy 3: Re-thought Nebuilder For HspA1, restriction sites NcoI and BbvCI could have been applied for cut & paste cloning Cutting close to the end of a sequence can be tricky, as some restriction enzymes are rather stringent PCR

Thank you Moral of the Story When it comes to cloning, there’s a plethora of different strategies to choose from No definite success can be guaranteed with any given method Develop yourself a thick skin and a hard head, (re)think both in and outside the box, and most importantly, don’t give up! Thank you

Questions? University of Helsinki Jaakko Itkonen Tania Quirin Manlio Fusciello Maarja Laos