igem2013 Microbiology BMB SDU

Transcription

1 igem2013 Microbiology BMB SDU Project type: BioBrick Project title: DXS biobrick from B. subtilis#168 Sub project: Creation date: Written by: Hwj Performed by: Hwj, MH, AK, PRA, SIS 1. SOPs in use igem2013_sop0010_v01_phusion PCR SDM: PCR reactions for Site directed mutagenesis of Dxs from B. subtilis igem2013_sop0014_v01_gel purification igem2013_sop0012_v01_restiction_digest igem2013_sop0019_v01_ Plasmid Miniprep 2. Purpose To create a biobrick with Dxs from B. subtilis 3. Overview Day SOPs Persons Experiments 15 SDM MH, AK, HWJ, PRA Performing site directed mutagenesis to remove EcoRI restriction site at 1065b in Dxs from B. subtilis # SDM PRA, AK Performing site directed mutagenesis to remove EcoRI restriction site at 1724b in Dxs from B. subtilis #168 Performing site directed mutagenesis to remove PstI restriction site at 1124b in Dxs from B. subtilis #168 Page 1 of 28

2 17 SDM PRA, AK Performing site directed mutagenesis to remove PstI restriction site at 1124b in Dxs from B. subtilis #168 Performing site directed mutagenesis to remove PstI restriction site at 1571b in Dxs from B. subtilis # SDM SOP0010 SOP0014 SOP0013 SOP0017 SOP0015 SOP SOP0021 SOP0014 SOP0017 SOP0019 PRA, SIS PRA, SIS SIS SIS SIS SIS PRA PRA SIS SIS MH Do site directed mutagenesis to remove PstI restriction site at 1571b in Dxs from B. subtilis #168 again, in order to get more sample. WIth the same purpose a Phusion PCR reaction, using sample 59 as template, was performed. Gel purification Nanodrop Fast digest on gel purification sample green 60 with EcoR1 and Pst1. Ligation of B.sub dxs (sample: red 62) and psb1c3 (sample red 58). Transformation. Colony PCR for B.sub dxs on transformed cells. Gel purification Fast digest with EcoR1 and Pst1 Miniprep on two cultures 20 MH, MHK Preparation for sequencing 21 SOP0010 SOP0012 SOP0015 PRA PRA PRA Phusion PCR with correct reverse primer to turn the suffix in the right orientation Digest of PCR product (Dxs) to be ligated into psb1c3 Ligation of EcoRI and PstI digested IspG into psb1c3 22 SOP0009 SF TSB Transformation 23 SOP0021 SIS, MH Colony PCR with Mytaq using VF2 and VR primere in order to check the size of the insert in psb1c3. 23 SOP0021 MH Repeat of PCR with Mytaq using VF2 and VR to check size of the insert in psb1c3 24 SOP0019 MH Plasmid miniprep on ONC in order to make a sample for sequencing. Sample prepared for sequencing. 25 PRA Prepared for shipping Page 2 of 28

3 4. Materials required Materials in use Name Components (Concentrations) Manufacturer / Cat. # Room Safety considerations primer Primers 002 and µM Sigma aldrich igem fridge 10µM Sigma aldrich igem fridge Primer µM Sigma aldrich igem fridge 5. Other comments 6. Experiment history Date (YY.MM.DD) SOPs Alterations to SOPs and remarks to experiments Page 3 of 28

4 SDM PCR reaction 1 and 2 in the site directed mutagenesis of Dxs B.subtilis was performed. The two purified templates from these PCR reactions were mixed in one tube. The PCR program: 50 deg 23 sec 30 cycles. 4 portions of 500 µl were prepared from the PHUSION SOP for each PCR reaction. For the 3 rd PCR reaction 100µl were prepared. PCR was run at 50 o C with 1 min elongation time and 30 cycles. Another PCR with the premixed template was conducted with 2.5µl template. 10µl total volume in each at 47, 50, 52 and 57 o C. 47, 52 and 57 were 40 cycles and 50 was 30 cycles. The PCR program: 55 deg 15 sec Page 4 of 28

5 SDM The expected band was not visible on the UV-table (see above, and results), and for this reason, a PCR reaction 1 and 2 were redone, although only 2 portions of 500 µl were prepared. The PCR program: 50 deg 23 sec 30 cycles. The purified products were not mixed, and these were used as template for the 3rd PCR reaction. The third PCR reaction were made with 5 different concentrations of template: 0.1, 0.5, 1.0, 2.0 and 5.0 µl. The PCR program: 55 deg 20 sec 35 cycles Page 5 of 28

6 Restriction digest SDM Grøn 44 and 45 were digested with EcoRI and checked on a gel that the removal of the EcoRI at 1065b was successful The fourth and fifth PCR reaction were each made with two different amounts of template (0.1µl and a pipette tip only dipped in Grøn 44). 20µl total reaction. PCR program: 55 deg 15 sec 30 cycles The sixth PCR reaction were made with 4 different amounts of each template (2.8, 14, 28 and 56 ng of each) PCR program: 55 deg 15 sec 35 cycles Restriction digest SDM Grøn 49 and 50 were digested with EcoRI and checked on a gel that the removal of the EcoRI site at 1724b was successful The seventh and eighth PCR reaction were each made with 0.1µl Grøn 49 (template). 25µl total reaction. PCR program: 55 deg 15 sec 30 cycles The ninth PCR reaction were each made with 1.5 µl Grøn 51 and 1 µl Grøn µl total reaction. PCR program: 55 deg 15 sec 35 cycles Page 6 of 28

7 Restriction digest SDM Grøn 53 and 54 were digested with PstI and checked on a gel that the removal of the PstI site at 1124b was successful The tenth and eleventh PCR reaction were each made with 0.1µl Grøn 53 (template). 25µl total reaction. PCR program: 55 deg 15 sec 32 cycles 72 deg 45 sec The twelfth PCR reaction were made with 0.5µl Grøn 56 and 1.0µl Grøn µl total reaction. PCR program: 55 deg 15 sec 35 cycles 72 deg 45 sec The twelfth PCR reaction were made with 0.5µl Grøn 56 and 1.5µl Grøn µl total reaction. PCR program: 55 deg 15 sec 35 cycles 72 deg 45 sec The twelfth PCR reaction were made with 0.5µl Grøn 56 and 1.5µl Grøn µl total reaction. PCR program: 50, 53, 57 and 60 deg 15 sec 35 cycles Restriction digest PCR 15µl Grøn 57 was digested with PstI and EcoRI and checked on a gel that the removal of the restriction sites was successful. 6µl psb1c3 was digested with the same restriction enzymes. Phusion PCR on Rød 59 (digested Dxs without restriction sites) PCR program: 60, 63, 67 and 70 deg 15 sec 30 cycles Page 7 of 28

8 Page 8 of 28

9 PCR SDM SOP0014 SOP0013 Phusion PCR on Rød 59 (digested Dxs without restriction sites) PCR program: 55, 59, 65 and 70 deg 15 sec 35 cycles The twelfth PCR reaction were made with 0.5µl Grøn 56 and 1.5µl Grøn µl total reaction. PCR program: 55, 59, 65 and 70 deg 15 sec 35 cycles Eluded with 30 µl water. incubation time with water: 5 min. SOP0017 Fast digest with EcoR1 and Pst1 on sample green 60: 5 µl DNA 3 µl buffer 1 µl of each enzyme (total 2 µl) 20 µl water Reaction time: 30 min. SOP0015 Ligation of B.sub dxs (sample: red 62) and psb1c3 (sample: red 58). 3 ligation reactions was performed: 1:2 2,5 µl plasmid 2 µl B.sub dxs 2 µl buffer 1 µl enzyme 12,5 µl water 1:5 2,5 µl plasmid 5 µl B.sub dxs 2 µl buffer 1 µl enzyme 9,5 µl water Negativ control 2,5 µl plasmid Page 9 of 28

10 2 µl buffer 1 µl enzyme 14,5 µl water RT for 2h and 45 min Transformation 5mL OD600=0.4 spun down Resuspended in 200µl TSB buffer 15µl ligation added 30 min on ice 2 min at 42 o C 1 h at 47 o C Plated on chloramphenicol plates Page 10 of 28

11 Colony PCR Fast digest One colony from the control plates was taken, and four from the plates with Dxs insert. 10µl MyTaq total reaction. PCR program: 95 deg 2 min 95 deg 10 sec 55 deg 15 sec 30 cycles 72 deg 30 sec Fast digest with EcoRI and PstI on colony PCR samples. The four fast digest samples: Colony 2: 13 µl DNA 3 µl buffer 1 µl of each enzyme 12 µl water Colony 3: 25 µl DNA 3 µl buffer 1 µl of each enzyme Colony 4: 7,5 µl DNA 3 µl buffer 1 µl of each enzyme 17,5 µl water Colony 5: 25 µl DNA 3 µl buffer 1 µl of each enzyme Digestion time: 30 min. Miniprep Colony 3 and 4 were minipreped after 6.5h of incubation at 37 o C and 160 rpm Sequencing 3 samples each containing 14 µl of sample 68, psb1c3- DXS (B. sub.). One sample contained 1µL primer 004, another 1µL 005 and the last one 1µL primer 019. Page 11 of 28

12 Phusion PCR Phusion PCR Restriction digest Ligation SOP Transformation Blue 67 were used as template in 25µL total reaction phusion PCR with primer 002 and 037 PCR program: 51, 53, 55.5, 57 deg 15 sec 30 cycles Blue 67 were used as template in 25µL total reaction phusion PCR with primer 002 and 037 PCR program: 55 deg 30 sec 5 cycles 65 deg 15 sec 30 cycles Green 74 was digested with EcoRI and PstI for 30 min at 37 o C Red 76 was ligated into psb1c3. 10 fmol psb1c3 to 20 and 50 fmol Red 76. The ligation was left at 16 o C overnight Two transformations - Plasmid:Red76 1:2 and 1:5. The TSB buffer was premade. 0,95mL culture and 10 µl plasmid were used. Phenotpical expression for 1 hr SOP0021 Colony PCR with Mytaq was performed on two colonies from the transformation plate 1:2 and 1:5. Annealing temp: 60 deg. Elongation time 30 sec. A mastermix containing water, primere (004 and 005) and Mytaq HS Red Mix was performed SOP0021 Colony PCR with Mytaq was performed on two colonies from each of the transformation plates 1:2 (13 & 14)and 1:5 (15 & 16). Annealing temp: 60 deg. Elongation time 30 sec. The samples were mixed in the PCR tubes and primers 4 and 5 were used SOP0019 Miniprep was performed using 200 µl water for elution Sample was prepared for sequencing using 14 µl of Blue 88 mixed with 1 µl of either primer 4, 5 or 19. Sample sent to sequencing. Page 12 of 28

13 ul of a concentration of 25ng/uL of Blue 88 was prepared for shipping. PCR tube#2 7. Sample specification Sample name Sample content Concentration Used for / Saved where Green 37 Green 40 Green 41 PCR products from PCRreaction1 and 2 with induced mutations at b.1067 of Dxs. PCR products using primer 2 and 15 PCR products using primer 2 and 15 to run PCR 3 to create a full Dxs without restriction site at 1065b. stored in the green box in the igem fridge 31.6 ng/µl to run PCR 3 to create a full Dxs without restriction site at 1065b. stored in the green box in the igem fridge 29 ng/µl to run PCR 3 to create a full Dxs without restriction site at 1065b. stored in the green box in the igem fridge Page 13 of 28

14 Green 42 Green 43 Green 44 Green 45 Green 46 Green 47 Green 48 Green 49 Green 50 PCR products using primer 3 and 14 PCR products using primer 3 and 14 PCR product with EcoRI at 1065b removed PCR product with EcoRI at 1065b removed PCR products using primer 2 and 21 PCR products using primer 3 and 20 PCR products using primer 3 and 20 PCR product with EcoRI at 1065 and 1724 b removed PCR product with EcoRI at 1065 and 1724 b removed 24.8 ng/µl to run PCR 3 to create a full Dxs without restriction site at 1065b. stored in the green box in the igem fridge 31.6 ng/µl to run PCR 3 to create a full Dxs without restriction site at 1065b. stored in the green box in the igem fridge 20.0 ng/µl To run PCR 4 and 5 for further removal of restriction sites. Stored in igem fridge 18.5 ng/µl To run PCR 4 and 5 for further removal of restriction sites. Stored in igem fridge 7.1 ng/µl to run PCR 6 to create a full Dxs without restriction site at 1724b. Stored in the green box in the igem fridge 14.2 ng/µl to run PCR 6 to create a full Dxs without restriction site at 1724b. Stored in the green box in the igem fridge 19.7 ng/µl to run PCR 6 to create a full Dxs without restriction site at 1724b. Stored in the green box in the igem fridge 11.9 ng/µl To run PCR 7 and 8 for further removal of restriction sites. Stored in igem fridge 9.0 ng/µl To run PCR 7 and 8 for further removal of restriction sites. Stored in igem fridge Page 14 of 28

15 Green 51 Green 52 Green 53 Green 54 Green 55 Green 56 Green 57 Red 58 Red 59 Green 60 PCR products using primer 2 and 17 PCR products using primer 3 and 16 PCR product with EcoRI at 1065 and 1724 b and PstI at 1124b removed PCR product with EcoRI at 1065 and 1724 b and PstI at 1124b removed PCR products using primer 2 and 19 PCR products using primer 3 and 18 PCR product with EcoRI at 1065 and 1724 b and PstI at 1124b removed PstI and EcoRI digested psb1c3 PstI and EcoRI digested SDM Dxs B.sub dxs without restriction sites. PCR on samples 55 and 56 mixed. Primer: 002 and ng/µl to run PCR 6 to create a full Dxs without restriction site at 1724b. Stored in the green box in the igem fridge 34.6 ng/µl to run PCR 6 to create a full Dxs without restriction site at 1724b. Stored in the green box in the igem fridge 32.4 ng/µl To run PCR 10 and 11 for further removal of restriction site. Stored in igem fridge 25 ng/µl To run PCR 10 and 11 for further removal of restriction site. Stored in igem fridge 8.2 ng/µl to run PCR 12 to create a full Dxs without restriction sites. Stored in the green box in the igem fridge 22 ng/µl to run PCR 12 to create a full Dxs without restriction sites. Stored in the green box in the igem fridge 19.7 ng/µl To be digested and inserted into psb1c3. Stored in igem fridge 5.6 ng/µl To be used for cloning. Stored in igem fridge 2.2 ng/µl To be inserted into psb1c3 and used as template for PCR ng/µl To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge. Page 15 of 28

16 Green 61 B.sub dxs without restriction sites. PCR on samples 59. Primer: 002 and ng/µl (not a very good graph) To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge. Red 62 Green 63 Green 64 Green 65 Green 66 Blue 67 Blue 68 Green 74 Red 76 B.sub dxs without restriction sites, digested with EcoR1 and Pst1 B. sub dxs without restriction sites. Colony PCR on colony 2 B. sub dxs without restriction sites. Colony PCR on colony 3 B. sub dxs without restriction sites. Colony PCR on colony 4 B. sub dxs without restriction sites. Colony PCR on colony 5 Miniprep of psb1c3- Dxs_B.sub for from colony 3 Miniprep of psb1c3- Dxs_B.sub for from colony 4 Phusion PCR product of B. sub Dxs with correct turned suffix. EcoRI and PstI digested Green ng/µl To be inserted into psb1c3 and used as template for PCR ng/µl To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge. 8.0 ng/µl To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge ng/µl To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge. 6.0 ng/µl To be used for digestion with EcoR1 and Pst1. Stored in the green box in the igem fridge. 58 ng/µl To be sequenced Stored in the green box in the igem fridge. 84 ng/µl To be sequenced. Stored in the green box in the igem fridge ng/µl To be digested and inserted into psb1c3. Stored in the green box in the igem fridge. 5.5 ng/µl To be inserted into psb1c3. Stored in the green box in the igem fridge. Blue 88 psb1c3-dxs (b. sub) 69.7 Plasmid with mutated Dxs from B. Sub. 8. Remarks on setup Page 16 of 28

17 9. Results and conclusions µl was loaded in each well on a 1% agarose gel, with ladder: 100 bp plus. The bands around was not visible on the UV table, first when the picture was taken, the bands appeared. For this reason, the PCR reaction 3 step was performed again. New round! Another round of first and second PCR was conducted and the bands seen below was purified and further used as template for third PCR reaction. Page 17 of 28

18 Using different amounts of both templates (0.1, 0.5, 1.0, 2.0 and 5.0 µl) several bands were seen, and the strongest just below 2000bp were cut out and purified Restriction digest to check for successful removal of EcoRI at 1065: Page 18 of 28

19 Only two bands are visible and both at the right length (approximately 243 bp and 1719 bp) SDM removing the EcoRI site at 1724b: The fourth and fifth PCR was run on an 1% agarose gel The one band between 1500 and 2000bp have the right length as well as the two bands just above 200bp. The three bands were cut out and purified. Page 19 of 28

20 The Sixth PCR was run on an 1% agarose gel Using different amounts of both templates several bands were seen, and the strongest just below 2000bp were cut out and purified. Restriction digest to check for successful removal of EcoRI site at 1724b: Only one band is seen and the removal of the two EcoRI sites has been successful! Yeaha! SDM removing the PstI site at 1124b: Page 20 of 28

21 The two PCR two clear bands at the right lengths. They were cut out and purified. PCR 9 were run on an 1% agarose gel The clear bands just below 2000bp were cut out and purified Restriction digest to check for successful removal of PstI site at 1124b: Only two bands at approximately 1566 and 387b were seen and the removal of the PstI site at 1124 was a success! Page 21 of 28

22 SDM removing the PstI site at 1571b: PCR 10 and 11 were run on an 1% agarose gel The bands at approximately 1500 and 300bp were cut out and purified. PCR 12.1 was run on an 1% agarose gel No PCR product, the PCR was run again Page 22 of 28

23 PCR 12.2 was run on an 1% agarose gel No PCR product at the right length, the PCR was run again PCR 12.3 was run an on 1% agarose gel The slighter clear band around 2000bp was cut out and purified. Restriction digest with EcoRI and PstI of psb1c3 and SDM DXS: Three bands were seen (hard to see on photo), so the uncut gel purified PCR product was run on another gel. Here the same three bands were seen. The band at 2000bp was cut out and purified to be used as template for further another round of PCR to purify it better. Page 23 of 28

24 The PCR with the band at 2000bp seen above as template was run on a 1% agarose gel There was no PCR product, and the PCR was done again. Results for new PCR: Ladder: Red. 1 % agarose gel. 25 µl was loaded in each well. Well 2-5 in the upper line contains the PCR 12, well 2-5 in the lower line contains PCR on sample red 59 Bands around 2000 bp appeared in all wells. The bands were cut out and purified. The nanodrop result on the purification samples: 48,6 ng/µl for green 60 and 58,0 ng/µl for green 61. Result for fastdigest with EcoR1 and Pst1: Ladder: red. 30 µl was loaded in each well on a 1% agarose gel. Page 24 of 28

25 Band appeared around 2000 bp in both wells. The bands were cut out and purified. Nanodrop result: 14,1 ng/µl (sample red 62). This samples is used for ligation with psb1c3 (sample red 58) Colony PCR was run on a 0.7% agarose gel First lane is from the control plate and the next four are from plates with Dxs insert. All four colonies have the correct insert length in their psb1c3 plasmid. The Four bands just above 2000bp were cut out and purified. Nanodrop result: Concentration ng/µl 260/ /230 Colony 2 15,2 1,88 0,07 Colony 3 8,0 1,52 0,13 Colony 4 27,3 1,26 0,40 Page 25 of 28

26 Colony 5 6,0 1,73 0,12 Restriction digest of purified DNA from colony PCR with EcoRI and PstI All of the four digested samples had the right length and the four restriction sites have successfully been removed Phusion PCR with primer 002 and 037 The PCR failed and was done again Phusion PCR with primer 002 and 037 Page 26 of 28

27 The two bands around 2000bp were cut out and purified Transformation was successful Results for the colony PCR on transformations: 10 µl was loaded in each well. Ladder: red. Well 5-8 contains colony PCR of cells transformed with dxs (B. sub) in psb1c3. The bands should have been around 2200 bp since we used Vf2 and VR primers. This is not the case. The bands appeared around 500 bp 300 bp. The bands around 300 bp could be relegations. The colony PCR was performed again on new colonies. Results of second colony PCR: Page 27 of 28

28 SUCCES! The bands in well 2,3 and 5 are just over 2000 bp. What s in well 4 is not known Miniprep was performed and elution was done in 200 µl. The concentration was estimated at 69.7 ng/µl using nanodrop. 10. Appendices Page 28 of 28