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

2. 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

3. 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

4. The Destination

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

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

7. 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!

8. 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

9. Strategy 2: Overlap Extension PCR
25-35 PCR Cycles

10. 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

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

12. 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

13. 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!

14. 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

15. Strategy 3: Re-thought Nebuilder

16. Strategy 3: Assembly

17. 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

18. 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

19. 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

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