Mini-Prep of the ligations for untagged antigens, pET_804 (3) and pIG15_804 (3) (RE)

• Qiagen-kit
• eluted in 30 µl dH2O
• NanoDrop:

Digest of the ILS plasmids (RE)

• Bba_K823013 with SpeI and PstI-HF
• Bba_K823008 with SpeI and PstI-HF
• Bba_K823005 with SpeI and PstI-HF
• Bba_I13504 with XbaI and PstI-HF

• 1 µl of each restr. enzyme
• 5 µl CutSmart
• 10 µl DNA
• 33 µl dH2O
• incubated for 1 h at 37°C

analysis on 1 % agarose gel:

Digest of pET_804 and pIG15_804 (RE)

Both plasmids were digested with BamHI and HindIII (to insert other antigens) as well as HindIII and AflII (to insert the Halo-tag instead of Spy)

• 1 µl of each restr. enzyme
• 5 µl FD Green Buffer
• 14 µl DNA
• 33 µl dH2O
• incubated for 5 min at 37°C

analysis on 1 % agarose gel: DNA is totally destructed by the enzymes

Repetition of the digest with NEB enzymes (RE)

• incubated for 1 h at 37°C

analysis on 1 % agarose gel: digest worked and the backbones were extracted from the gel (TAE buffer was exchanged before running the gel, maybe it caused the problems with DNA destruction instead of the restriction enzymes)

Test digest of pET_SpyC_GST (RE)

• 1 µl HindIII
• 1 µl PstI-HF
• 2 µl CutSmart
• 5 µl DNA
• 11 µl dH2O
• incubated for 1 h at 37°C
• expected fragment sizes: ~1700 and ~4100 bp

Test digest of pET_x02 (untagged antigens) (RE)

• 1 µl BamHI
• 1 µl PstI-HF
• 2 µl CutSmart
• 5 µl DNA
• 11 µl dH2O
• incubated for 1 h at 37°C
• expected fragment sizes:

analysis of both test digests on 1 % agarose gel: DNA is destructed by the enzymes (TAE buffer was exchanged afterwards); only for pET_1502 and pET_1702, correct fragments are visible among the smeer

Ligations (RE)

• InterLab study:

• Halo-tag:

• Antigens:

• 1 µl T4 ligase
• 2 µl T4 ligase buffer
• up to 20 µl dH20
• incubated for 1 h at RT

Transformation of the ligation products (5 µl) into E.coli TOP10 (RE)

• pILS1-3, pIG15_805 and pIG15_1004-1704 were plated on LB-cml
• pET_1004-1704 were plated on LB_amp

Sequencing results (LS)

• pET_804 (3): correct
• pET_804 (4): correct
• pIG15_804 (3): correct
• pIG15_804 (4): correct

Test digest: pEtX04, pIG15_X04, pILS1-3 and pIG15_805 (LS)

• Interlab Study: pILS1, 2 and 3–>EcoRI HF and SpeI HF

• pETX04, pIG15_X04, pIG15_805–>BamHI HF and PstI HF

• addition of 5µl plasmid each
• incubation at 37°C, 1h

• analysis on 1% agarose gel:

Upper part: First six rows are the digested plasmids for the interlab study. pILS1 is pSB1C3 with Anderson Promoter 17 (J23117) followed by GFP (~ 940bp). pILS2 contains the Anderson Promoter 06 (J23106), and pILS3 the Anderson Promoter 01 (J23101). Test digest of the plasmids for protein expression (pETX04)in E.coli with His_tag-antigen-Spy_tag insert. Expected band length for the different constructs: pET_1004:~1900bp pET_1104:~2700bp pET_1504:~3100bp pET_1704:~1900bp Lower part: Test digest for the Cellfree Expression Constructs, containing a His_tag-antigen-Spy_tag insert. Expected band lenght for the different constructs: pIG15_1004:~800bp pIG15_1104:~1500bp pIG15_1504:~1900bp pIG15_1704:~800bp Test digest of pIG15_805 for cellfree expression containing His_tag-antigen 8-Halo_tag. Expected band lenght of insert: ~1500bp.

PCR for biobricks + PCR for GFP insert (LS)

Antigens 6, 8, 10, 11, 17 and 18 will be amplified using primers with matching overlap for pSB1C3 for Gibson cloning. The plasmids pIG15_X01 containg antigen fragments will be used as template. For GFP amplification pQE-60 GFP will be used as template.

+0,1µ template (~100ng/µl) +1µl of each primer

• analysis on 1% agarose gel:

Upper part:Fragment 6,10,11 and 17. Expected band lengths: fragment 6:312bp fragment 10:657bp fragment 11:1440bp fragment 17:624bp Lower part: Fragment 18 and GFP. Expected band lengths: fragment 18:615bp GFP:~800bp Antigen 6 could not be amplified and antigen 11 shows only a very slight band a the correct size. The other fragments were amplified correctly.

Gel-ex: PCR products (LS)

• qiagen kit

Digest: pIG15_104 with EcoRI HF and SpeI HF (LS)

For Gibson assembly of the biobricks containing the antigens, digested backbone is needed. pIG15_104 is the cellfree expression vector inside the pSB1C3 standard and can thus be used for digestion.

• 1h, 37°C
• analysis on 1% agarose gel
• gel-ex: 45,0ng/µl

Inoculation of Gly-Stocks in liquid LB-Amp (LS)

To build biobricks with all antigens some of the Gly-stocks have to be inoculated again, followed by miniprep of the respective plasmids. The following E.coli Gly-stocks were inoculated:

• pIG15_301
• PIG15_401
• pIG15_501
• pIG15_701
• pIG15_901

• incubation at 37°C, shaking

Gibson-Assembly (RE)

• inserts: PCR products of the antigens 8, 10, 11, 17 and 18
• backbone: pSB1C3, EcoRI and SpeI digested
• DNA-Mix:

• 5 µl were directly transformed into E. coli TOP10

Transformation (RE)

• pET_804
• pIG15_804
• pET_SpyC_GST
• pIG15_1104
• pILS_1
• pILS_2
• pILS_3
• pET_804 and pET_SpyC_GST were plated on LB_amp
• all the others were plated on LB_cml

Inocculation of 5 ml liquid culture (RE)

• pET_804 (LB_amp, 2x)
• pIG15_804 (LB_cml, 2x)
• pET_SpyC_GST (LB_amp)
• pIG15_1104 (LB_cml)
• pILS_1 (LB_cml)
• pILS_2 (LB_cml)
• pILS_3 (LB_cml)

Preparation of Gibson MasterMix (RE)

• according to the protocol of AG Weber
• 100 µl 5X ISO Buffer
• 50 µl Taq ligase
• 6.25 µl Phusion polymerase
• 0.2 µl T5 Exonuclease
• 218.55 µl ddH2O
• –> 15 µl aliquots stored at -20°C

Mini-Prep (LS)

• qiagen kit

PCR for amplification of the antigens (LS)

• amplification of the following antigens: 3,4,5,6,7,9
• 2×20µl each (antigen 6: 4×20µl)
• PCR mix and cycling protocol same way as last time (3.8.2015)

Inoculation (LS)

• 3 colonies from Gibson-Assembly plates each
• 5ml LB-Amp
• incubation at 37°C o/n

Mini-Prep (RE)

• Qiagen-Kit
• eluted in 30 µl dH2O
• pET_804
• pIG15_804
• pET_SpyC_GST
• pIG15_1104

Digest of pET_804 and pIG15_804 (RE)

• ~2 µg per construct were digested with BamHI and HindIII for insertion of antigens
• ~1 µg per construct was digested with HindIII and AflII for insertion of the Halo-tag
• 1 µl of each restriction enzyme (Fermentas)
• 5 µl FD Green Buffer
• ad 50 µl dH2O

Analysis of PCR (from yesterday) on 1% agarose gel (LS)

Antigen fragments 3,4 and 6 were amplified correctly. 5,7 and 9 did not work well. Expected band lengths: antigen 3: ~1100bp antigen 4: ~1100bp antigen 5: ~1100bp antigen 6: ~260bp antigen 7: ~1600bp antigen 9: ~1300bp

Gel-ex: PCR products for biobricks (LS)

• qiagen kit

Digest: pET804 with BamHI+HindIII and HindIII+AflII (LS)

• incubation at 37°C, 30 min

Mini-Prep of Gibson products for standardized plasmids (LS)

• Zymo Research kit

Test digest of standardized plasmids (LS)

• digest with: EcoRI HF and PstI HF

• incubation at 37°, 1h
• analysis on 1% agarose gel

Expected band lenghts for digested fragments: antigen 8:~430bp antigen 10:~600bp antigen 17:~580bp antigen 18:~570bp. For pRIG15_8, clone 3 shows correct bands, for pIRG15_10 clone 3, pRIG15_17 clone 1 and pRIG15_18 clone 2 and 3 are correct. Correct clones were sent for sequencing.

PCR for amplification of antigens 5, 7, 9 and 11 (RE)

• gradient for annealing temp., because the PCR did not work for those antigens the last time
• 5X Master-Mix per antigen
• 20 µl Phusion High GC Buffer
• 5 µl of each primer
• 2 µl dNTPs
• 3 µl DMSO
• 1 µl Phusion polymerase
• 0.5 µl Template (100 ng/µl)
• 63.5 µl dH2O

cycling:

• the same cycling conditions as before were used, except the gradient for the annealing temperature (55°C-65°C)

analysis on 1% agarose gel:

• amplification of 5, 7 and 9 was successful for every annealing temperature
• amplification of 11 did not work again

Gel-Ex of the PCR products (RE)

• Qiagen-Kit
• eluted in 20 µl dH2O
• 5: 72.4 ng/µl
• 7: 98.3 ng/µl
• 9: 76.7 ng/µl

Ligations (RE)

• 1 µl T4 ligase
• 2 µl T4 ligase buffer
• ad 20 µl dH2O
• incubation 1 h at RT
• transformation of 5 µl ligation product into E. coli TOP10

Gibson-Assembly (JN)

• assembly of pSB1C3 backbone with different antigens for standardization

Inoculation of ligation/gibson clones (LS)

• 2x each
• 5ml LB-Amp/Cml
• 37°C, shaking

Repetition of PCR for biobrick fragments: 10, 11, and 18 (LS)

• 4×20µl, gradient PCR

• analysis on 1% agarose gel
• 11 did not work…

Mini-Prep (JN)

• pRIG15_06/07, pIG15_1004/1504/1704, pET_1004/1104
• Qiagen kit
• eluted in 30µl dH2O

Mini-Prep (RE)

• Biozym-kit (Solution 2 from Peqlab-kit was used, because it was empty in the Biozym-kit)
• eluted in 30 µl dH2O
• pET_1504 - 1: 156.4 ng/µl
• pET_1505 - 2: 84.2 ng/µl
• pET_1704 - 1: 104.8 ng/µl
• pET_1704 - 2: 144.7 ng/µl
• pIG15_805 - 1: 189.1 ng/µl

Test Digests (RE)

• pRIG15_06 and pRIG15_07 with EcoRI-HF and PstI-HF (NEB –> 1 h incubation at 37°C)
• pET_1001, pET_1104, pET_1504, pET_1704, pIG15_1004, pIG15_1504, pIG15_1704 with BamHI and HindIII (Fermentas FD –> 30 min incubation at 37°C)
• pIG15_805 with HindIII and AflII (Fermentas FD –> 30 in incubation at 37°C)

Result of the test digests

Repetition of Test Digests (JN)

• pET_1001, pET_1104, pET_1504, pET_1704, pIG15_1004, pIG15_1504, pIG15_1704 with BamHI and HindIII (Fermentas FD –> 15 min incubation at 37°C)

Gel-ex: PCR product for biobricks 10 and 18 (LS)

• qiagen kit
• eluted in 20µl

Repetition of PCR for biobrick 11 (LS)

• 4×25µl

• analysis on 1% agarose gel

Transformation (LS)

• 5min, RT

• transformation of blunt-end ligation/ gibson (7µl) in E.coli T10
• transformation of pIG15_105 (1µl) in E.coli T10
• transformation according to protocol

PCR for fragment 17 with HA-tag (LS)

• analysis on 1% agarose gel
• gel-ex:qiagen kit

Inoculation: pIG15_105 and pIG15_805 (LS)

• 5ml LB-ml
• 37°C, shaking o/n

Inoculation: pRIG15_10, pRIG15_18, ligation of pJet_GFP (JN)

• 3 clones for each
• 5ml LB-medium (pRIG with Cml, pJet with Amp)
• 37°C, shaking, until evening

Gibson Assembly (JN)

• antigen 11 with pSB1C3 backbone to send it in
• pET_803 backbone with 1703-HA
• pET_804 backbone with 1704-HA

• transformation into E.coli Top10 and plating (pRIG on Cml, pET on Amp)

Mini-Prep of pIG15_105 and pIG15_805 (JN)

• Zymo Research kit
• eluted in 30 µl dH2O
• NanoDrop:

Digest: pIg15_105, pIG15_804 and pIG15_805 (LS)

• pIG15_105: HindIII + AflII
• pIG15_804:BamHI + HindIII
• pIg15_805: BamHI + HindIII
• 5µl FD green buffer
• 1µl of Enzyme 1 and 2 each
• up to 50µl dH2O
• incubation at 37°C for approx. 45min
• analysis on 1% agarose gel: no results for pIG15_105 and pIG15_805
• gel-ex (qiagen kit): pIg15_804 backbone. 65,8ng/µl

Blunt-end ligation: Antigen 15 (Salmonella) in pJET + Transformation (LS)

• 5min, RT
• transformation in E.coli T10 (transformation according to protocol)

Mini-prep of pJet_GFP and pRIG15_10 (RE)

• Zymo-kit
• pelleted cells were resuspended in LB medium
• elution in 30 µl dH2O
• NanoDrop:

Test digest of pJet_GFP and pRIG15_10 (RE)

• pJet_GFP was digested with BamHI and HindIII (FastDigest; 30 min incubation)
• pRIG15_10 was digested with EcoRI-HF and PstI-HF (NEB; 1 h incubation)
• 1 µl of each restriction enzyme
• 2 µl of the respective buffer
• 5 µl DNA
• 11 µl dH2O
• incubation at 37°C

Inocculation of liquid cultures (RE)

• pRIG15_11 (LB_cml)
• pET_1703_HA (LB_amp)
• pET_1704_HA (LB_amp)
• 3 clones each

Mini-Prep of pRIG15_11 (antigen 11 for iGEM Registry), pET_1703-HA and pET_1704-HA (JN)

• Zymo Research kit
• eluted in 30 µl dH2O
• NanoDrop:

Test digest of all clones above (LS)

• pRIG15_11. EcoRI + PstI (1h incubation at 37°C)
• pET1703/4-_HA: BamHI + AflII (30 min incubation at 37°C)
• 5µl plasmid
• 1µl of each restriction enzyme
• 2µl 10x buffer
• up to 20µl dH2O
• analysis on 1% agarose gel

Digest of pJet_GFP 1-3, pET_1703_HA and pET_1704_HA (RE)

• 1 µl BamHI
• 1 µl HindIII
• 5 µl FD Green Buffer
• 10 µl DNA
• 33 µl dH2O
• incubation for 30 min at 37°C

Ligation (LS)

• added 2µl T4 DNa Ligase buffer
• 1µl T4 DNA Ligase
• up to 20µl dH2O
• incubation at Rt for 1h
• Transformation into E.coli T10 (according to protocol)

Retrafo: pET_1703-HA in E.coli T10 (LS)

• 1µl plasmid
• transformation according to protocol

Inoculation of pSB1C3_SpyC (JN)

• 3 colonies were picked and inoculated in 5ml each of LB-medium with Cml

Mini-prep of pJet_15 (JN)

• ZymoResearch kit
• eluted in 30µl dH2O
• concentrations

Digest with BamHI and HindIII of pJet_15 (JN)

• 37°C for 30min
• analysis on 1% agarose gel

Expected fragment sizes: pJet backbone ~3000bp, fragment 15 ~1700bp. The bands were visible at the expected heights and the bands for fragment 15 were cut out

Gel-ex of fragment 15 (JN)

• fragment 15, digested with BamHI and HindIII, was extracted out of the gel
• Qiagen gel extraction kit
• concentrations

Inoculation of many colonies (RE/JN)

• Gibson and ligation
• in 5ml LB medium
• 2 colonies each

Mini-prep of pSB1C3_SpyC_correct (JN)

• ZymoResearch kit
• eluted in 30µl dH2O
• concentrations

Test-digest of pSB1C3_SpyC_correct (JN)

• 37°C for 1 hour
• analysis on 1% agarose gel

Mini-prep (JN)

• Qiagen kit
• eluted in 30dH2O
• concentrations

Test digests (RE)

• pRIG15_10

–> EcoRI + PstI

• pET_805
• pIG15_805

–> HindIII + AflII

• pET_1704
• pET_203_HA
• pET_1103_HA
• pET_1104_HA
• pET_1504
• pET_1004
• pET_1104
• pET_204
• pET_204_HA
• pIG15_1504
• pIG15_1704
• pIG15_204

–> BamHI + HindIII

Mini-Prep (LS)

• qiagen kit

Sequencing (RE)

• pET_203_HA - 1
• pET_204 - 1
• pET_204_HA - 1
• pET_1104 - 1
• pET_1104_HA - 1
• pET_1704 - 1
• pIG15_204_HA - 1
• pIG15_1704 - 1
• pIG15_805 - 1
• pRIG15_10 - 2

edit 08/13: all results are positive, except pRIG15_10. pIG15_805 should be sequenced with a reverse primer again to verfiy the whole Halo-tag sequence.

Ligations (RE)

• 1 µl T4 ligase
• 2 µl T4 ligase buffer
• incubated for 1h at RT
• directly transformed into E. coli TOP10 (pET constructs plated on LB_amp; pIG15 constructs plated on LB_cml)

Inocculation of liquid cultures (RE)

• yesterday's ligations
• 2 single colonies per construct

Test digest: pIG15_1704 (LS)

• 5µl plasmid
• 1µl BamHI + HindIII
• 2µl buffer
• up to 20µl dH2O
• analysis on 1% agarose gel

Inoculation of pIG15_1504/10004, pET_1004/805/1504 (JN)

• 5ml LB-medium each

Mini-Prep of pIG15_1704-HA (JN/LS)

• Zymo Research kit
• eluted in 30µl H2O
• concentrations

Test-Digest pIG15_1704-HA (LS)

Mini-Prep of pIG15_1504/10004, pET_1004/805/1504 (JN)

• Zymo Research kit
• eluted in 30µl H2O
• concentrations

Digest of pIG15_805 (RE)

• 1 µl BamHI
• 1 µl HindIII
• 5 µl FD Green Buffer
• 5 µl DNA
• 38 µl dH2O
• incubation for 30 min at 37°C

Gel-Ex of pIG15_805 backbone (RE)

• Qiagen-kit
• eluted in 20 µl dH2O
• NanoDrop: 39.6 ng/µl

Ligations (RE)

- Backbone: pIG15_805 (BamHI and HindIII digested; 39.6 ng/µl) 1.25 µl
- Inserts:

• fragment 10: 0.87 µl
• fragment 11: 0.9 µl
• fragment 15: 3.78 µl
• fragment 17: 2.14 µl
• GFP: 1.67 µl
• + ligation control

- Backbone: pET_804 (HindIII and AflII digested) 0.86 µl
- Insert: Halo-tag 0.66 µl

• incubation for 1 h at RT
• 5 µl ligation product were directly transformed into E. coli TOP10

Transformation for ProtPur (RE)

• pET_203_HA into E. coli BL21 pLys
• pET_1104_HA into E. coli C43
• pET_1704_HA into E. coli BL21 pLys

Mini-Prep of Retrafo pET_1703-HA (JN)

• Zymo Research kit
• eluted in 30µl dH2O
• concentration: 57.1ng/µl

Test-Digest of pET_805/1004/1504, pIG15_1004/1504 (JN)

• 30min at 37°C
• analysis on 1% agarose gel but with unknown ladder –> no reliable statement about digest possible

expected fragment sizes: pET_1004 ~570+5500bp, pET_1504 ~1700+5500bp, pET_805 ~940+5900bp

expected fragment sizes: pIG15_1004 ~570+2400bp, pIG15_1504 ~1700+2400bp

Digest (RE)

• analysis on 1% agarose gel

expected bands were visible and cut out pJet_15: ~1700+3000bp pET_1104: 1400+5500bp pET_1704-HA: 540+5500bp

expected bands were visible and cut out pIG15_804: ~400+2400bp pIG15_805: ~400+3300bp pIG15_1704-HA: ~540+2400bp

Gel-ex of fragment 15, backbones pET_1104/1704-HA, pIG15_804/805/1704-HA (JN)

• Qiagen kit
• eluted in 20µl dH2O

Ligation of fragment 15 into different backbones, GFP into pIG15_805, Halo into pET_804 (JN)

• 1 µl T4 ligase
• 2 µl T4 ligase buffer
• incubated for 1h at RT

Transformation (RE)

• 5 µl of the ligation products and the Gibson Mix into E. coli TOP10
• pET constructs were plated on LB_amp
• pIG15 constructs were plated on LB_cml

Mini-prep (RE)

• Zymo-Kit; elution into 30 µl dH2O
• pIG15_1005 - 1: 230.9 ng/µl
• pIG15_1005 - 2: 162.1 ng/µl
• pIG15_1105 - 1: 121.3 ng/µl
• pIG15_1105 - 2: 117.8 ng/µl
• pIG15_1505 - 1: 96.6 ng/µl
• pIG15_1505 - 2: 139.1 ng/µl

Test digests (RE)

• pIG15_1005, pIG15_1105 and pIG15_1505 with BamHI and HindIII (FastDigest)
• pRIG15_10 with EcoRI and SpeI (NEB)
• 0.5 µg DNA
• 1 µl of each RE
• 2 µl of the respective buffer
• ad 20 µl dH2O
• incubation for 30 min (FastDigest) or 1 h (NEB) at 37°C

Seuencing (RE) The following constructs have been send in for sequencing:

• pIG15_1005 - 2
• pIG15_1105 - 1
• pIG15_1505 - 1
• pIG15_805 - 2 (again with the pET_RP to verfiy the sequence of the Halo-tag)

Re-trafo (RE)

• of pIG15_1704_HA into E. coli TOP10

Inoculation of pET_1504/1504-HA, pIG15_1504/1504-HA/1505/1705-HA/205 (JN)

• 2 single colonies were picked of each plate
• in 5ml LB medium (pET with Amp, pIG15 with Cml)

Resuspension of IDT gBlocks (JN)

• pOP fragment 1 was eluted in 50µl dH2O, pOP fragment 2/3/4/5 were eluted in 100µl dH2O after short centrifugation
• according to the protocol delivered by IDT the fragments were incubated at 50°C for 20min and afterwards vortexed and centrifuged again

Ligation of pOP fragments into blunt end pJet (JN)

• the delivered fragments were cloned into blunt end pJet according to protocol

• incubation for 5min at RT
• directly afterwards trafo with 5 µl according to protocol into E.coli TOP10

Gibson Assembly 1 of pOP (JN)

• pOP fragments 2+3+4 were assembled via Gibson according to protocol

• trafo with 5µl according to protocol into E.coli TOP10 followed directly after short incubation

Mini-Prep (JN)

• Zymo Research kit
• eluted in 30µl dH2O

Inoculation (JN)

• pJet_pOP fragments in 5ml LB-Amp
• Re-trafo pIG15_1704-HA in LB Cml
• Gibson 1 pOP in LB without antibiotics

Test-digest of pET_1504/1504-HA, pIG15_1504/1504-HA/1505/1705-HA/205 (JN)

• all prepared the same way with BamHI and HindIII

• incubation for 30min at 37°C
• analysis on 1% agarose gel

expected fragment sizes: pET_1504 ~1700+5500bp pET_1504-HA ~1700+5500bp pIG15_1504 ~1700+2400bp pIG15_1504-HA ~1700+2400bp

expected fragment sizes: pIG15_1504-HA ~1700+2400bp pIG15_1505 ~1700+3200bp pIG15_1705-HA ~540+3200bp pIG15_205 ~740+3200bp

Mini-Prep of pOP Gibson 1 (JN)

• Qiagen kit
• eluted in 30µl dH2O

Digest of pOP Gibson 1 (8) and (10) (JN)

• 30min at 37°C
• analysis on 1% agarose gel

expected fragment size: ~3400 bp. Nothing was visible at all –> no positive clones were picked

Digest of fragment 2 + 4 of pOP for 5-fragment Gibson (JN)

• fragment 2 digested with HindIII
• fragment 4 digested with BamHI
• 30min at 37°C
• analysis on 1% agarose gel

expected fragment sizes: ~1300bp for fragment 2, ~800bp for fragment 4. In both cases, strangely two bands were visible. Anyways, the upper bands were considered correct and extracted from the gel.

Gel-ex fragment 2+4 digest (RE)

• Qiagen-Kit
• eluted in 15 µl dH2O
• fragment 2: 6.7 ng/µl
• fragment 4: 8.1 ng/µl

Gibson assembly for pOP (JN)

• fragment 1, digested fragment 2, fragment 3, digested fragment 4, fragment 5

• DNA added to Gibson Master Mix
• after 5min of incubation at RT, 5µl were used for a transformation into E.coli TOP10 according to protocol
• the rest was incubated for 1h at 50°C, 3min at RT, 3min on ice
• then another transformation was performed
• both were plated on Amp plates

Mini-Prep of pJet_pOP fragments and pIG15_1704-HA (JN)

• Zymo Research kit
• eluted in 30µl dH2O

PCR for pOP Gibson (JN)

As the fragments for the pOP vector were wrongly cloned, there is a antibiotic resistance missing after the first Gibson Assembly. Therefore, colonies cannot be selected for taking up the plasmid. Yesterday we tried an approach with a 5 fragment Gibson Assembly but no colonies were growing on the plate overnight. The next approach is to assemble two and accordingly 3 fragments via PCR. We decided to use fragments 5, 1 and 2 for one reaction, where the antibiotic resistance would be restored. The other reaction should assemble fragments 3 and 4. The PCR reaction mixes were prepared as follows. For the first reactions, the primers pOOP_5fw and pOOP_2rv were used, for the second pOOP_3fw and pOOP_4rv. As template a 1:10 dilution of the prepped pJet constructs (1) from yesterday was prepared. From this dilution 1µl per fragment was used.

• analysis on 1% agarose gel

expected fragment sizes: pOP_521 ~3000bp –> the bright band was excised pOP_34 ~2100bp –> the faint lower band was excised

Gel-ex of pOP_512 and pOP_34 (JN)

• Qiagen kit
• eluted in 20µl dH2O

Gibson Assembly of PCR products pOP_512 and pOP_34 for pOP (JN)

• 1.5µl of pOP_512 and 3.5µl of pOP_34 were added to Gibson Master Mix
• after 5min of incubation at RT, 5µl were used for a transformation into E.coli TOP10 according to protocol
• the rest was incubated for 1h at 50°C, 3min at RT, 3min on ice
• then another transformation was performed
• both were plated on Amp plates

PCR for SpyCatcher amplification (RE)

• The SpyCatcher was amplified with two different reverse primers. oIG15_SpyC_02 a HindIII restrcition site, oIG15_SpyC_03 a BamHI site. PCR fragments obtained by 02 are named 1-2, the others 1-3.

Cycling (25 cycles):

Digest of pET_SpyC and pET_SpyC_GST (RE)

• digests to open the backbone for insertion of the real SpyC sequence (wrong sequence is excised)
• pET_SpyC was digested with BamHI and HindIII
• pET_SpyC_GST was digested with BamHI
• 1 µl of each RE
• 5 µl FD Green Buffer
• 1 µg DNA
• 1 µl Antarctic phosphatase
• ad 50 µl dH2O

Analysis of the SpyC PCR products and the backbone digests (RE)

• 1 % agarose gel
• both amplifications were successful
• both digests were successful
• all fragments were extracted from the gel
• Qiagen-kit
• elution in 30 µl dH2O
• NanoDrop:

Digest of the purified SpyCatcher PCR products (RE)

• 1-2 was digested with BamHI and HindIII
• 1-3 was digested with BamHI
• 1 µl of each RE
• 5 µl FD Green Buffer
• 17 µl DNA
• 26 µl dH2O
• incubated for 30 min at 37°C

Gel-Ex (RE)

• of the digested SpyC PCR products
• Qiagen-kit
• eluted in 20 µl dH2O

Blunt end ligation of the SpyCatcher PCR products (undigested!) into pJET (RE)

• 1 µl pJet
• 10 µl reaction buffer
• 1.5 µl PCR product
• 1 µl T4 ligase
• 6.5 µl dH2O
• incubated for 5 min at RT and subsequently transformed into E. coli TOP10 (plated on LB_amp)

Transformation (RE)

• pOP 2-fragment Gibson (50°C incubation) after fusion PCR (JN)
• E. coli TOP10; plated on LB_amp

Ligations (SpyCatcher) (RE)

1.:

• pET_SpyC (BamHI / HindIII digested): 1.61 µl
• PCR product 1-2 (same digest): 0.52 µl
• dH2O: 14.87 µl
• T4 ligase: 1 µl
• T4 ligase buffer: 2 µl

2.:

• pET_SpyC_GST (BamHI digested): 3.33 µl
• PCR product 1-2 (same digest): 0.42 µl
• dH2O: 13.25 µl
• T4 ligase: 1 µl
• T4 ligase buffer: 2 µl

Incubated at 16°C over night

Transformation: Ligation pET_Spy-C (LS)

• 7µl of ligation mix
• transformation according to protocol
• incubation on LB-Amp plates at 37°C

Miniprep: pOP and pIG15_1504-HA (LS)

• qiagen kit

Picked clones from ligation plate (LS)

• 3x pET_SpyC
• 3x pET_SpyC-GST
• incubation in 5ml LB-Amp at 37°C o/n

Pelleting of liquid culure (LS)

• 5ml liquid culture
• pJET-Spy-C
• frozen at -20°C

Mini-Prep (RE)

• 6 cultures pelleted yesterday by LS (pJet_SpyC 1-2; pJet_SpyC 1-3)
• 3 cultures of pET_SpyC (ligation after PCR product digest)
• 3 cultures of pET_SpyC_GST (ligation after PCR product digest)

(Test)Digest (RE)

• pET_SpyC_GST and pJet_SpyC 1-3 with BamHI
• pET_SpyC and pJet_SpyC 1-2 with BamHI and HindIII
• pOP with PstI and MluI
• DNA: 1 µg for pJets; 0.5 µg for pET and pOP
• Buffer: 5 µl CutSmart for pOP; FD Green for the others
• 1 µl of each RE
• ad 50 µl dH2O
• incubation for 1 h (pOP) or 30 min at 37°C

Dilution of primers (LS)

• biobrick SpyC (GST)
• Salmonella mutagenesis

PCR: Salmonella mutagenesis (LS)

• first part: oIG15_1503f + oIG15_1504r
• second part: oIG15_1505f + oIG15_1506r
• third part: oIG15_1507f + oIG15_1508r

Gel-ex: Spy-Catcher (LS)

1. Spy-C: BamHI digested 2. Spy-C: BamHI and HindIII digested

• qiagen kit:

Fusions PCR for pOOP fragments (RE)
Fragment 5-1:

Fragment 2-3-4:

Cycling (25 cycles):

Gel-ex: PCR product for Salmonella mutagenesis (LS)

• qiagen kit
• eluted in 20µl

Digest of pIG15_104 to get pSB1C3 backbone (RE)

• 10 µl DNA
• 5 µl CutSmart
• 1 µl XbaI
• 1 µl PstI-HF
• 33 µl dH2O
• incubation for 1 h at 37°C

Ligation: Spy-Catcher (LS)

• backbone: pET_SpyC(old) BamHI or BamHI+HindIII digested
• insert: gel-ex of SpyC (GST) fragment

• 1h, RT

Transformation: Ligation of pET_SpyC (-GST) into E.coli T10 (LS)

• 10µl of ligation mix
• transformation according to protocol
• plated on LB-Amp
• incubation at 37°C o/n

Picking colonies from pET_SpyC (-GST) Ligation (LS)

• 3x 5ml LB-Amp each
• incubation at37°C

Gel-ex: pSB1C3 XbaI and PstI digested (LS)

• qiagen kit
• concentration (20µl): 14,3ng/µl

Fusion-PCR: pOOP (LS)

• analysis on 1%agarose gel
• fusion of frg. 1+5 worked, fusion of frg. 2+3+4 did not work

Gibson: Salmonella mutagenesis (LS)

• added 15µl Gibson mix
• incubation at RT, 5 min
• transformation in E.coli T10
• plated on LB-Cml

Gel-ex of fusion PCR product 5-1 (RE)

• qiagen kit
• eluted in 20µl dH2O
• NanoDrop: 11.8ng/µl

Repetition of Fusion PCR (RE)

• assembly of pOP fragments 2 and 3

Cycling (4 cycles):

Addition of Primers oIG15_pOOP2_fw and oIG15_pOOP3_rv (2.5 µl each)

Cycling (20 cycles):

PCR clean-up of fragment 2-3 (RE)

• Qiagen-kit
• elution in 20 µl dH2O
• NanoDrop: 7.2 µl

Fusion PCR of frg. 2-3 and 4 (RE)

^ Ingredient ^ Volume [µl] ^

Cycling (4 cycles):

Addition of Primers oIG15_pOOP2_fw and oIG15_pOOP4_rv (2.5 µl each)

Cycling (20 cycles):

Mini-Prep (RE)

• pET_SpyC (3x; ligation with pJet digest product)
• pET_SpyC_GST (3x; ligation with pJet digest product)
• Zymo-kit
• elution in 30 µl dH2O

Test-Digest of pET_SpyC and pET_SpyC-GST (RE)

• 8 µl DNA
• 2 µl FD Green Buffer
• 1 µl BamHI
• (1 µl HindIII - only for pET_SpyC)
• ad 20 µl dH2O
• incubation for 30 min at 37°C

Repetition of Fusion PCR (LS)

• same components/cycling as last time
• 2×25µl

Cycling (4 cycles):

Addition of Primers oIG15_pOOP2_fw and oIG15_pOOP3_rv (1µl each)

Cycling (20 cycles):

• analysis on 1% agarose gel
• correct band at ~2700bp visible
• gel-ex (qiagen kit): 20µl –>18.5ng/µl

• second PCR: fusion of frg. 4 to frg.2-3 (2×25µl)

Cycling (4 cycles):

Addition of Primers oIG15_pOOP2_fw and oIG15_pOOP4_rv (1µl µl each)

Cycling (20 cycles):

• analysis on 1% agarose gel
• correct band at ~3400bp visible
• excision/ frozen at -20°C

Picking clones from Gibson: Salmonella mutagenesis (LS)

Mini-prep of pRIG15_15 (RE)

• Zymo-Kit
• eluted in 30 µl dH2O
• pRIG15_15 - 1: 54.9 ng/µl
• pRIG15_15 - 2: 47.2 ng/µl
• pRIG15_15 - 3: 37.8 ng/µl

Gel-Ex of Fusion PCR product 2-3-4 (RE)

• Qiagen-Kit
• eluted in 20 µl dH2O
• NanoDrop: 7.4 ng/µl

Test digest of pRIG15_15 (RE)

• 7 µl DNA
• 1 µl EcoRI-HF
• 1 µl SpeI
• 2 µl CutSmart
• 9 µl dH2O
• incubation for 1 h at 37°C

Mini-Prep of pOOP (3x) (RE)

Colony PCR (LS)

• 12 colonies from ligation plate + control (pSB1C3 with Spy-Catcher from munich)
• dipped sterile filter tip in colony, put some of the colony in PCR tube, streaked the rest of th colony on new LB-Amp plate

• analysis on 1%agarose gel

PCR for Salmonella antibody (JN)

• 2×25µl

• analysis on 1% agarose gel
• no bands were visible at all

Repetition: PCR for Salmonella antibody (LS)

• 2×25µl

• analysis on 1% agarose gel
• correct bands visible: gel-ex (qiagen kit)

Gibson assembly for pRIG15_13 (LS)

• 2,13 µl pSB1C3 (PstI+XbaI)
• 0,74µl frg. 13
• added to Gibson mix
• 5 min, RT
• transformation into E.coli Top 10

Repetition of transformation of E.coli Top 10 with Gibson mix for pRIG15_13 (LS)

Miniprep of pET_SpyC 8-12 (RE)

Picked one clone from Gibson plate for pRIG15_13 (LS)

Repetition of PCR for frg. 13 (LS)

• same as last tim
• analysis on 1% agarose gel: correct
• gel-ex (qiagen): 138,3ng/µl

Miniprep: pRIG15_13 (1) (LS)

• 5ml
• Zymo kit: 216,5 ng/µl

Test Digest: pRIG15_13(1) with EcoRI HF and SpeI HF (LS)

• 37°C, 1h
• analysis on 1% agarose gel: not convincing

Repetition of Gibson assembly for pRIG15_13 (LS)

• 2,03µl pSB1C3 (XbaI + PstI)
• 0,60µl frg. 13
• added Gibson MasterMix
• 5min, RT
• transformation into E.coli Top 10

Transformation: pOP (2) + (LS)

• 2µl plasmid
• transformation according to protocol

Digest of pOP, BBa_I712669 and BBa_K592009 (JN)

• analysis on 1% agarose gel, correct fragments for pOP and BBa_I712669 were cut out of the gel

Gel-ex of pOP and BBa_I712669 (JN)

• Qiagen kit
• eluted in 20µl dH2O

Ligation of pOP and BBa_I712669 (JN)

• 1h at RT

Trafo of ligation product (10µl) into E.coli TOP10 (JN)

• plated on LB-Amp plates