igem2013 Microbiology BMB SDU

Transcription

1 igem2013 Microbiology BMB SDU Project type: Conventional cloning of IspG together with LacI-PLac-dxs(B.sub) in psb1c3. Project title: LacI-PLac-dxs(B.sub) in psb1c3 Sub project: Creation date: Written by: SIS, MHK Performed by: SIS, HWJ, MHK 1. SOPs in use igem2013_sop0017_v01_ Fast digest igem2013_sop0014_v01_ Gel purification igem2013_sop0015_v01_ Ligation igem2013_sop0009_v01_ TSB transformation igem2013_sop0021_v01_colony PCR SOP0019_Miniprep 2. Purpose Cloning of IspG together with LacI-PLac-dxs(B.sub) in psb1c3. 3. Overview Day SOPs Persons Experiments 1 SOP0017 SOP0014 SIS SF Fast digest of blue 167 (psb1c3-laci-plac-dxs(b.sub)) with EcoRI and Xbal and blue 87 (IspG) with EcoRI and Spe. Gelpurification of digested psb1c3-laci-plac-dxs(b.sub) and IspG Ligation of red 176 and red 177 SOP0015 Hwj 2 SOP0009 SIS, MH Transformation of ligations into MG SOP0021 SIS Colony PCR on transformations from with VR and VF primere.

2 3 SOP0021 SF Colony PCR on transformations from with VR and VF primere. 4 SOP0017 SOP0015 HWJ HWJ Digestion of blue 167 with SpeI and PstI and Fast AP Digestion of blue 87 with PstI and EcoRI Ligation 1:0, 1:2, 1:5 5 SOP0021 HWJ Mytaq colony PCR 6 ONC MHK ONC of colonies from 30/9 7 SOP0019 HWJ, SF Miniprep of overnight cultures with colonies #20-30 and #32 8 SOP0017 SIS Test digestion of plasmid purified from colony 30 with EcoRI and PstI in order to check if the insert has the right size (4519 bp). 9 SOP0017 SIS Test digestion of plasmid purified from colony 38, 50 and 51 with EcoRI and PstI in order to check if the insert has the right length (1116 bp). 9 SOP0017 SOP0014 SOP0017 SOP0015 MH SIS SF SF PRA Digestion of plasmid from colony 38, 50 and 51 with EcoRI alone in order to linearize the plasmid for comparison with test digestion. Digestion of blue 167 with Spel and PstI and blue 87 with PstI and Xbal. Gelpurification of digest of blue 167 and blue 87 Fastdigest of Blue 87 with PstI and Xbal. Ligation of red 209 and 211 (should become psb1c3-laci-placdxs(b.sub)-ispg) 10 SOP0009 Hwj Transformation into E.coli MG1655 with 1,5 hour phenotypical expression 11 SOP0021 HWJ, MH SF Mytaq colony PCR 2 times New digest of blue 167 with SpeI, PstI and Fast AP Gel purification Ligation 12 SOP0009 SF Transformation into E. coli KG22 13 HWJ ONC of colony #10 from SOP0019 SOP0017 SOP0021 MHK HWJ Miniprep of ONC colony 10 Fast digest test with EcoRI and PstI of colony 10 miniprep Colony PCR 15 SOP0021 HWJ Mytaq colony PCR SOP0019 SOP0017 MHK MHK Miniprep of ONC of colony #31 Test fast digest with E+P and E of miniprep 19? SOP0009 MHK, HWJ TSB transformation of psb1c3-laci-dxs(b)-ispg into MG SOP0021 SIS Mytaq colony PCR 21 SOP0019 SF, Hwj Plasmid miniprep Test digest

3 4. Materials required Materials in use Name Components (Concentrations) Manufacturer / Cat. # Room Safety considerations 5. Other comments 6. Experiment history Date SOPs (YY.MM.DD) Fast digest Gelpurification Ligation TSB transformation Alterations to SOPs and remarks to experiments Blue 167 was digested with EcoRI and Xbal for 20 min (not 5 min) and Fast AP was added to the digestion mixture. Blue 87 was digested with EcoRI and SpeI also for 20 min. 5 ul blue 167 was used and 14 ul blue 87. The SOP was followed and nanodrop was measured. Ligations of red 176 and red 177 were prepared acording to the SOP. Three ligations were prepared with: 10 fmol plasmid and 0, 20 and 50 fmol insert. The three ligations (1:0, 1:2 and 1:5) were transformed in MG1655. Expression time: 1 hour.

4 Colony PCR Colony PCR Colony PCR Fast digest Ligation Mytaq colony PCR was performed on four colonies from each of the transformation plates 1:0 (colony 9), 1:2 (colony 1-4) and 1:5(colony 5-8) with VR and VF primere. Mytaq colony PCR was performed on four colonies from each of the transformation plates 1:0 (colony?), 1:2 (colony 1-4) and 1:5(colony 5-8) with VR and VF primere. Mytaq colony PCR was performed on four colonies from each of the transformation plates 1:0 (colony 23), 1:2 (colony 24-27) and 1:5(colony 28-31) with VR and VF primere. Digestion of blue 167 with SpeI and PstI and Fast AP Digestion of blue 87 with PstI and EcoRI Run on gel and purified, stored as samples: Red 186 (psb1c3-plac-dxs) with a concentration of 13,6 ng/µl Red 186 (IspG) with a concentration of 5,6 ng/µl Ligation 10fmol red186 to 20 and 50 fmol red 187 Transformation Transformation followed SOP, with phenotypical expression for 2 hours Colony PCR Mytaq colony PCR was performed on four colonies from each of the transformation plates 1:0 (colony 9), 1:2 (colony5-8) and 1:5(colony 1-4) with VR and VF primere. Colony PCR was made again: Mytaq colony PCR was performed on four colonies from each of the transformation plates 1:0 (colony 36), 1:2 (colony32-35) and 1:5(colony28-31) with VR and VF primere ONC Over night cultures were made of colonies 28, 29, 30 and 32 from the colony PCR 30/ ml of LB was added to each Miniprep Miniprep of overnight cultures with #28-30 and # 32 samples stored as blue Fast digest Sample blue 192 was digested with EcoRI and PstI for 20 min. 30,6 ul DNA was used. Total volumen: 34,6 ul. And 192 was digested with EcoRI alone in order to linearize the plasmid.

5 Fast digest Gelpurification Digestion of blue 167 with Spel and PstI and blue 87 with PstI and Xbal. No phosphatase was added, unfortunately. The gel was purified - the dillution was done with 100 µl Fastdigest water by accident. Blue 87 with X+P. The sop was followed. Ligation Ligation 10 fmol red209 to 0, 20 and 50 fmol red Transformation Transformation into E.coli MG1655 with 1 hour phenotypical expression Mytaq colony PCR Fast Digest Gel purification Ligation TSB transformation Plasmid miniprep Fast digest Colony PCR of transformations from the 6/9 Colony PCR of transformations from the 6/9 Colony PCR: Mytaq colony PCR was performed on 9 colonies from the transformation plates 1:0 (colony 1), 1:2 (colony2-5) and 1:5(colony6-9) with VR and VF primere. Colony PCR was made again: Mytaq colony PCR was performed on 16 colonies from with VR and VF primere. Fastdigest of Blue 167 with SpeI and PstI with Fast AP The sop was folowed. Nanodrop was measured. The ligations were made with 10 fmol psb1c3-laci-placdxs(b. sub) and 0, 20 and 50 fmol IspG and left overnight at 16 degrees. The SOP was followed, 1 hour phenotypical expression. Plated on LA plates with CM. 3 tubes of approximately 1,5mL of the ONC from (colony 10 from transformation ) were miniprepped and pooled. The sample from above (blue 220) was digested with EcoRI and PstI for 20 minutes. Colony PCR: Mytaq colony PCR was performed on 9 colonies from the transformation plates 1:0 (colony 18), 1:2 (colony10-13) and 1:5(colony 14-17) with VR and VF primere. Colony PCR: Mytaq colony PCR was performed on 9 colonies from the transformation plates 1:0 (colony 26), 1:2 (colony27-29) and 1:5(colony 30-32) with VR and VF primere.

6 Miniprep Miniprep of ONC of colony #31 from minipreps were done and pooled. Fast digest Test digest of miniprep (Blue 225) with E+P and E. Digested according to SOP (5min at 37 deg C) TSB TSB transformation of psb1c3-laci(natural)-dxs(b)-ispg transformation 1:0, 1:2 and 1:5 into MG1655. Expression time 1h Colony PCR Mytaq colony PCR with VR and VF primers was performed on four colonies from transformation plate 1:2 (colony 13-16), four colonies from transformation plate 1:5 (colony 17-20) and transformation plate 1:0 (coloni 21) Plasmid miniprep Miniprep of colony nr from transformation d. 15/9 with correct lenght. 10 ml ONC was used. The SOP was followed. Test digest Test digest of colonies #18 and#19 7. Sample specification Sample name Sample content From/concentration Used for / Saved where Red 176 psb1c3-laci-plac- Blue 167 igem fridge DXS(b.sub) dig. with E+X. WRONG! Red 177 IspG digested with E+S Blue 177 igem fridge WRONG! Blue psb1c3-laci-plac- igem fridge DXS(b.sub) - IspG WRONG! Red 209 psb1c3-laci-placdxs(b.sub) Blue 192/5,7 ng/µl igem fridge digested with S+P Red 217 psb1c3-laci-placdxs(b.sub) Blue 167 igem fridge digested with S+P+phosphatase Blue 225 psb1c3-laci-plac-dxs Miniprep of colony 31 igem fridge (B)-IspG from 7/9, 30,0 ng/µl Blue 248 psb1c3-laci(no LVA)- Plac-DXS (B)-IspG Miniprep of colony 18 fro 15/9: 42,5 igme fridge

7 Blue 249 psb1c3-laci(no LVA)- Plac-DXS (B)-IspG Miniprep of colony 19 fro 15/9: 40,5 igme fridge 8. Remarks on setup 9. Results and conclusions Result for fast digestion: 20 ul was loaded on a 1% agarose gel with red ladder. Well two contains psb1c3-laci-plac-dxs(b.sub) digested with E and X. Well three contains IspG digested with E and S and well four contains psb1c3-lacdxs(e.coli) digested with E and X. In well two band of the right length (5400 bp) appeared. In well three band with the right length also appeared (1200 bp). The bands were cut out and purified. Nanodrop of gelpurification: psb1c3-laci-plac-dxs(b.sub): 5,6 ng/ul IspG: 4,4 ng/ul Result for colony PCR: No bands appeared so the colony PCR was performed again. Results for new colony PCR: Well loads: Green ladder, colony 23, colony 24-27, green ladder, colony The colonies contains plasmids with ligations of the following ratios (psb1c3-laci-plac-dxs (B.)-ISPG) Colony 23 1:0 Colony :2 Colony :5

8 Unfortunately the wrong digestion enzymes were chosen. The plasmid (blue 167) should have been digested with Spe and PstI and ISPG (blue 87) should have been digested with EcoRI and PstI, so the cloning have to be redone. Fucking fuck pis! The first mytaq colony PCR showed no correct bands, however a lot of the wells showed nothing, so a new PCR reaction was made First PCR: well 2-10 (colonies 1-9) expected bands 4300bp Second PCR:well 2-10 (colonies 28-36) expected bands 4300bp : Miniprep of colonies psb1c3-laci-plac-dxs(sub)-ispg gave the condentrations: 26,5, 21,0, 32,7 adn 52,7 ng/µl.

9 Test digestion result: 1% agarose gel, ladder: red. Digestion of colony 38 is loaded in well three, of colony 50 in well four and of colony 51 in well five. Two bands appeared. One around 2000 bp, which corresponds to the plasmid, and one around 1200 bp which indicates that the the digestion unfortunately has cut out the insert in the plasmid (LacI-PLacdxs(B.sub)) so only the ISPG has been inserted. Going back in the protocol revealed that the insert has been cut with PstI and EcoRI but should have been cut with PstI and Xbal. So the digestion was performed again Nanodrop of digests: Red 209: 5,7 ng/µl Red 210: 0,4 ng/µl Since this concentration is so small, the ISPG (Blue 87) was cut again: Digest of blue blue ladder. 6 is digest. The lower band (1200 bp) was cut out and purified giving a new sample: Red 211 with concentration 8,3 ng/µl

10 First colony PCR showed 2 bands on all but one colonies including the religation. 2 nd colony PCR Fast digest of blue 167 with pst and spe gave nice bands around 5400bp. was cut out and purified with a concentration of 7,2 ng/µl Miniprep produced sample Blue 220 with a nanodrop of 63 ng/µl.

11 Fast digest/test digest of sample Blue 220 was loaded in well number 2.. Red ladder in well number 1. The band between 2000 and 2500bp is expected to be the plasmid backbone. The other band was expedted to be 4300bp, but was approximately 3500bp. Colony PCR with Mytaq: There is no band in the religation well, and no bands with the correct length #26-32 in well 9 is the religation (#26) Plasmid miniprep

12 Blue 225 was stored with a concentration of 30,0 ng/µl. Test digest Well load: Blue 225, Blue 225 cut with EcoRI, Blue 225 cut with EcoRI and PstI, green ladder No bands of correct size. In well number 2 a band appeared a bit above 5000bp, the expected size was. In well number 3 a band appeared a bit above 3000bp and 2000bp, the expected sizes were approximately 2000bp and 4300bp Result for the colony PCR with mytaq and VR and VF primers: 10 µl was loaded in each well. Ladder: red. Colony are LacI(without LVA taq?)-plac-dxs(b.sub)-ispg. Colony 21 is a negative control. Bands should have appeared around 4000 bp, but three appeared 2000 bp and two around 1000 bp. Unfortunately no band appeared for the negative control, so we performed an ONC od colony 18 and 19 in order to check the insert sizes with a test digestion Miniprep on colony 18 and 19 see blue 248 and 249 in table.

13 Test digest of colonies #18 and#19 showed that laci-plac-dxs has been cut out and IspG is the only insert. (bad picture see real one in the folder) 10. Appendices