igem2013 Microbiology BMB SDU

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1 igem2013 Microbiology BMB SDU Project type: Biobrick Project title: DXS biobrick from B. subtilis Sub project: Creation date: Written by: HWJ, PRA & SF Performed by: HWJ, ASF, SIS, MHK, SF & PRA 1. SOPs in use igem2013_sop0009_v01_ TSB transformation igem2013_sop0010_v01_phusion PCR igem2013_sop0012_v01_restriction_digest igem2013_sop0013_v01_nano_drop igem2013_sop0014_v01_gel purification igem2013_sop0015_v01_ ligation igem2013_sop0001_v01_mm_on_culture_of_e.coli igem2013_sop0017_v01_ Fast digest igem2013_sop0019_v01_plasmid Miniprep igem2013_sop0021_v01_tk_colony_pcr_with_mytaq 2. Purpose To create a biobrick with Dxs from B. subtilis Page 1 of 18

2 3. Overview Day SOPs Persons Experiments 1 SOP0010 HWJ & PRA Colony PCR with Phusion on B. sub with primers 002 and SOP0013 SOP0012 SOP0014 HWJ, PRA, SF & MH Gel purification and nanodrop PCR on part of the purified DNA Digestion of purified DNA Digestion of plasmid backbone Gel purification and nanodrop of cut DNA Ligation 3 SOP0009 PRA, MHK & SF 4 SOP0010 SF, SIS & MH Transformation Phusion PCR on B. Sub with primers 002 and SOP0012 SOP0013 SOP0014 SOP0015 SOP0001 SF, HWJ, MHK Gel purification Nanodrop Digest of purified DNA Digest of plasmid backbone Gel purification of digested DXS and Plasmid backbone Ligation of DXS and plasmid backbone ON of E. coli for transformation 6 SOP0009 SF, HWJ, MHK, MH, SIS Transformation 7 SOP0017 HWJ, MH digestion of DXS with PstI and EcoRI 8 SOP0019 SOP0021 SOP SOP0021 SOP0010 SOP0019 SOP SOP0017 SOP0015 SOP0019 MHK, SIS HWJ, MHK, SIS, MH HWJ, MHK SIS, MH Plasmid Mini prep on RFP-pSB1C3 plasmid Colony PCR of too short DXS from B. Subtilis 3x Colony PCR with phusion polymerase of B. Subtilis DXS Plasmid Mini prep on DXS (B.subtilis) -psb1c3 plasmid. Frozen colony of transformed E.coli cells with shortened B.subtilis DXS. Digest test for PstI, EcoRI, XbaI, BamHI. Digest + ligation of psb1c3 + DXS (B. Sub.) with XbaI and BamHI RFP-pSB1C3 plasmid mini prep 11 SOP0009 HWJ, MHK Transformation with of E.coli with ligations of DXS and psb1c3, cut with BamHI and XbaI. Page 2 of 18

3 12 SOP0019 SOP0017 MH, MHK, HWJ Miniprep of an ON culture of E.coli with psb1c3 including RFP digestion of psb1c3 including RFP with XbaI and BamHI. Note: The igem standard restriction enzymes are EcoRI, XbaI, SpeI and PstI, not BamHI. 13 SOP0017 HWJ, SIS, Kir Test digestion of B.subtilis with EcoRI and PstI Sequence analysis of B. subtilis 168 in sequence massager. Primers for site directed mutagenesis have been designed and ordered primers SOP0021 SOP0010 Hwj Colony PCR with phusion polymerase of B. Subtilis #168 DXS 4. Materials required. Materials in use Name Components (Concentrations) Manufacturer / Cat. # Room Safety considerations B. subtilis N/A Primer µm Sigma Aldrich igem fridge N/A primer µm Sigma Aldrich igem fridge N/A Gel/PCR product from plasmid backbone psb1c3 igem N/A N/A Mini prep kit igem storage N/A Chlorampheni col 5. Other comments The igem plasmid psb1c3 contains restriction sites for EcoRI, XbaI, SpeI and PstI, not BamHI. Page 3 of 18

4 6. Experiment history Date (YY.MM.DD) SOPs Alterations to SOPs and remarks to experiments SOP0010 A small amount of B. subtilis colony was used as template Annealing temperature: 51 o C Elongation time: 2 min 10 sec Cycles : SOP0010 A small amount of B. subtilis colony was used as template Annealing temperature: 51 o C Elongation time: 2 min 10 sec Cycles: SOP µl of lineraized Plasmid backbone was used. The restrictionenzymes was EcoRI og PSTI. 10 µl of water was used SOP µl of purified gel containing the DXS gene was used. The restrictionenzymes was EcoRI og PSTI. No water was used SOP0014 All purified DNA from gel was used SOP0013 Nanodrop, digested DSX: Solvent used was water SOP fmol plasmid and 0, 10 and 20 fmol PCR product was used. This was 0, 4 and 8 µl (calculated from Nanodrop results). Overnight at 16 deg C SOP0009 Scince the Photospectrometer didn t cooperate we didn t measure OD : We aded 15 µl Plasmid with the concentrations of 0, 10 and 20 fmol to three eppendorftubes SOP0010 A mastermix of 7x were prepared. we made 6 50µL samples but didn t do a gradient. The concentration of the DNA template is unknown: we only had one drop left. The temperature of segment 2.2 were 47,8, segment 2.3 lasted 1 minutes. Page 4 of 18

5 SOP0014 We made 3 samples. 1) the content of 1 well. 2) the content of 2 wells. 3) the content of 3 wells. We added 100µL capture buffer pr. 0,10g gel: 170µL in sample 1, 440µL in sample 2 and 400µL in sample 3. The spin was at x g. We did the flow through the columns two times in different ependorftubes SOP0013 We did a blank in between each sample. Sample 1 from the first elution had a concentration We pooled the the samples from the second spin of the gelpurification SOP times DXS from B. Subtilis were cut in each an Eppendorf tube. 15µL of 76,2ng/µL of B. Subtilis DXS was used. 10µL of plasmid psb1c3. Enzyme 1 was EcoRI, enzyme 2 was PstI SOP µl of capture buffer 3 was added to 0,130 g of gel with plasmid (both cut and uncut) PCR product. 125 µl of capture buffer 3 was added to 0,120 g of gel with DXS B. Subtilis PCR product. Regarding point 8.6 in the SOP: 30µL of water was added to the column and spun down in the tube in stead of the flow (meaning that the volume of the purified DNA solution was 60µL) SOP0015 Ligation of the plasmid backbone and DXS was done with 4 different ratios 1:5, 1:10 and 1:20 and a negative control with 1:0 with no DXS. Due to an error (see point 8.2)with the purification no water was added to the ligation mixture. For the 1:5 a volume of 12,5 μl DXS and 4,5 μl plasmid. 1:10 a volume of 14,5 μl DXS and 2,5 μl plasmid. and finally for 1:20 a volume of 15,5 μl DXS and 1,5 μl plasmid. 5 μl plasmid was added to the negative control. The samples has been placed on 16 o C ON in the basement. Page 5 of 18

6 SOP0009_v preparation of E.coli culture: 277 μl ONC was added to 50 ml LB media in a 250 conical flask in order to get a OD600=0.05, but it was OD600=0,028. It was decided that this was sufficient. After 1.5 hours growth the OD600 was measured every 30 min until OD600= TSB transformation: plasmid amount: 10 μl of our own ligation and 1 μl of igem RFP plasmid (BBa_J04450). Transformation overview: Dxs-plasmid 1:0 (x2) Dxs-plasmid 1:5 (x2) Dxs-plasmid 1:10 (x2) Dxs-plasmid 1:20 (x2) igem RFP plasmid 0,5 pg/μl (x2) igem RFP plasmid 50 pg/μl (x2) Ie. a total of 12 transformations. Phenotypical expression at 37 deg. for 1 hour. Transformation plates are placed in the growth room at 37 deg over night SOP0017_v01 To check which fast digest enzymes cut DXS at an unexpected restriction site, we made 4 digestions: 1 st EcoRI for 5 min, 2 nd EcoRI for 20 min, 3 rd PstI for 5 min and 4 th with PstI for 20 min. See remarks on setup 8.3. the gel was loaded in the order 1,2,3, SOP0019_v01 SOP0013_v01 10,3µL chloramphenicol-solution of 17,5 mg/ml was added to 5 ml LB. A colony of RFP-pSB1C3 containing E. Coli cells was transferred to the LB and put in the heating cabinet for 2½h. The final OD 600 was measured to be ml of this was transferred to each 3 Eppendorf tubes since 3 minipreps were to be done (3mLs total). After the miniprep, the concentrations of DNA obtained in each of the samples were 2.7, 2.3 and 1,8 ng/µl (measured with nanodrop). The samples were pooled and one Eppendorf tube with a concentration of 2.2 ng/ µl of the RFP-pSB1C3 plasmid is stored in the fridge. Page 6 of 18

7 SOP0021_v01 The colony used from the transformation of psb1c3-dxs with the ratio 1:5 (5x DXS to 1x plasmid). 8 samples were prepared. Point 8.1 of the SOP was done before point 8.1. After having transferred a colony from the plate to the Eppendorf tube the rest of the colony was transferred to another plate. This was to be able to identify the different samples. Point 8.5 of the SOP was not done. 0,5µL template was added to 9,5µL master mix. The annealing temperature was 60 deg C SOP0021_v01 SOP0010_v01 SOP0013_v01 SOP0014_v SOP0019 SOP0013 SOP SOP0017_v01 Colony PCR was made from B. sub to get DXS. Step of SOP0021 was done, then SOP0010 was followed. 10x master mix was prepared, but the phusion polymerase was not added until the solution was in the PCR tubes. 8 reactions with 20µl were used. T m was 52,8. Only a small band was visible this was purified and 2 new reaction was prepared in the same way, but the polymerase was now added to the mastermix one with 8x20µl and one 8x50µl. The master mix with 50µL was vortexed for <2 seconds and quickly spun down (after the polymerase was added). They were run on a gel and purified. Nanodrop was measured on all the samples. The concentration of the Mini prep samples were estimated using Nanodrop. A frozen culture was prepared by mixing 750 µl ON culture with 250 µl 50% glycerol in autoclaved freezing tubes. Fast digest was done with PstI, EcoRI, XbaI and BamHI. The DNA used was DXS from B. Subtilis (purified 02/ 07-13). By mistake a MgCl solution was used instead of water. A second fast digest was performed as above, except water was used as stated in the SOP. PstI and EcoRI both had restriction sites within the B. Subtilis DXS gene. SOP0019_v01 Mini prep on RFP-pSB1C3 plasmid containing E.coli cells (ONC) Page 7 of 18

8 SOP0017_v01 SOP0014_v01 SOP0015_v01 Fast digest was done with XbaI and BamHI on DXS from B. Subtilis and psb1c3 containing RFP. The cut DXS and plasmid were cut from a gel and purified according to the SOP. Ligation of the plasmid backbone and DXS was done with 3 different ratios 1:3, 1:5 and 1:10 and a negative control with 1:0 with no DXS. 1,61 µl plasmid was used in all ligations. V DXS varied as follows: 1:3 4,80 µl 1:5 8,05 µl 1:10 16,10 µl The samples have been placed on 16 o C ON in the basement SOP0009_v01 Transformation of E.coli with 10µl of the ligations from : plasmid:dxs 1:0, 1:3, 1:5, 1:10, pure E. coli and E. Coli with psb1c3 containing RFP (0,5pg/µl and 50pg/µl). The chloramphenicol containing plates with E. Coli were put in the 37 deg C heating cabinet SOP0021 SOP0017 Miniprep on ON from E. coli with psb1c3 Digestion on psb1c3 obtained from miniprep. Digestion was with XbaI and BamHI for 10 min instead of 5. Two fast digests were done. In the first one, an unknown, but too big, amount of water was added. The second one was, except for digestion time, done according to the SOP SOP0017 Digestion of B. Sub with EcoRI and PstI. And sequence ( seqmassager.htm) Checking for restictionsites for EcoRI, PstI, SpeI and XbaI Primers for site directed mutagenesis have been designed and ordered. (Primers ) SOP0021 SOP0010 Colony PCR with phusion polymerase of B. Subtilis#168 DXS Mixtures prepared for sequencing with a 5 ng/µl concentration and 1 µl primers: Tube: 002 Primer: 004 Tube: 003 Primer: 005 Tube: 004 Primer: 019 Page 8 of 18

9 7. Sample specification Sample name Sample content Description Used for / Saved where Blue 100: RFP-pSB1C3 Blue 101: Dxs-psB1C3 Red 1: Truncated Dxs in E.coli The psb1c3 containing an RFP gene. Too short Dxs (B.subtilis) in psb1c3 plasmid E.coli cells containing plasmids with too short B.subtilis Dxs Backbone with RFP gene, used for sending BioBricks to igem/saved in the red igem box in the freezer. Saved in the red IGEM box in the freezer Saved in the -80 freezer green 9 B. subtilis DXS (1,3ng/µl) Stored in the green box in the fridge green 10 B. subtilis DXS (46ng/µl) Stored in the green box in the fridge Green 11 B. subtilis DXS (3,9ng/µl) Stored in the green box in the fridge Page 9 of 18

10 green 12 B. subtilis DXS (37ng/µl) stored in the green box in the fridge Blue 13 RFP-pSB1C3 plasmid concentration: 19,2 ng/μl Blue 14 RFP-pSB1C3 plasmid concentration 24,3 ng/μl stored in the green box in the fridge stored in the green box in the fridge Green 30 B. subtilis DXS (22,9ng/ µl) Stored in the green box in the fridge Green 31 B. subtilis DXS (38ng/µl) Stored in the green box in the fridge 8. Remarks on setup 8. On the gel with digested DXS a band at the length of 500 bases was visible and was not expected to be there. The expected length of DXS from B. subtilis was 1922 bases long and was slightly less than 1500b. Acording to Nebcutter there is no restriction site in the gene. 8. During the purification there was eluted with 60μl H 2 O instead of 30μl when we checked for restriction sites in DXS we forgot to put a ladder in the gel. No band was visible for the digestion with PstI 5 min. 9. Results and conclusions The gel was 1,0% agarose and the light blue (100bp plus) ladder was used. The PCR product was loaded in well number 3. There was a small band just below 2000 bp, which was cut out for purification. The expected length was 1945 bp. Page 10 of 18

11 Gel purification yielded a concentration of 4ng/ul measured by Nanodrop. Gel purification of digested Plasmid and DXS gave 5,6 and 3,2 ng/µl No results of the transformation Page 11 of 18

12 All 50 µl of each of the six samples were loaded in a 1% agarose gel with the light blue ladder. The bands of DSX was cut out and put in tree ependorfubes - the content of 1 well in one tube, 2 ells in another and 3 in the last Nanodrop results of the DXS purifications: 1 band from gel 2 bands from gel 3 bands fro gel 1. flow through 21,3 ng/µl 74,8 ng/µl 76,2 ng/µl 2. flow through 4,5 ng/µl 13,6 ng/µl 13,4 ng/µl Gel from PCR of cut plasmid and DXs: Page 12 of 18

13 Cut plasmid in well 2, uncut plasmid in 3, uncut DXS in 4, cut DSX in 5 and 6 ladder in 1 and bp to much cut from DXS The transformations was only successful for the DXS-plasmid 1:5 and igem RFP plasmid 50 pg/µl. The agar plates was in general too wet, so the colonies were indistinguishable For all 8 colony PCRs a band at 1500 bp appeared, so all colonies contains plasmids with a fragment of B.subtilis dxs. Result for the mini prep: purified psb1c3 plasmid containing RFP with concentration of 2.2 ng/µl was stored in the green igem box in the fridge. The test digestion with EcoRI indicated that there could be a restriction site 500bp in DXS from the B. subtilus. Page 13 of 18

14 The concentration of the Miniprep sample was 29,4 ng/µl. Colony PCR results The first 8 samples were not very clear on the gel, possibly because they were not mixed properly before the PCR run. 1 band was purified and yielded a solution with 1,3ng/µl stored as sample Green 9 in the green box(fridge). The other 16 samples were run on a gel after the PCR, 13 lanes were purified and nanodrop was measured. Some of the purifications were pooled and stored in the green box. See sample specification 7 Green Page 14 of 18

15 Restriction test digest results Test digestion of B. subtilis DXS with all igem standard restriction enzymes showed that EcoRI and PstI both cut within the B. Subtilis DXS gene. BamHI and XbaI did not. The digests were loaded in the following gel: Page 15 of 18

16 Ligation The samples have been placed on 16 o C ON in the basement. Results will be noted tomorrow. Mini prep result 1) 19,2 ng/µl 2) 24,3 ng/µl Transformation with of E.coli with ligations of DXS and psb1c3, cut with BamHI and XbaI, was done. There was no positive colonies, all colonies on the plates appear as if RFP is still present. This suggests that digestion of psb1c3 was not successful It was discovered that the psb1c3 plasmid does not contain the BamHI restriction site, but rather the SpeI. Page 16 of 18

17 INSERT PICTURE 4 Ladder: 100bp plus. Second well and third well: psb1c3 cut with XbaI and BamHI. Since there is no restriction site for BamHI in the psb1c3 plasmid, the RFP gene was not cut out. The band seen at approximately 3000 bp is believed to be psb1c3 with RFP (~2700bp). INSERT picture 5 Ladder: 100bp plus. Second well: psb1c3 cut with XbaI and BamHI. Since there is no restriction site for BamHI in the psb1c3 plasmid, the RFP gene was not cut out. The band seen at approximately 3000 bp is believed to be psb1c3 with RFP (~2700bp). A sample of uncut psb1c3 was loaded in the third well, however there was a loading error, and the contents did not sink to the bottom of the well. They are not on the picture The digestion test showed that both PstI and EcoRI had 2 restictionsites. Running the sequence in showed that there is 2 restrictionsites for EcoRI and PstI and none for SpeI and XbaI Colony PCR 2 samples were stored Green 30 with 22,9ng/µl and green 31 38ng/µl Page 17 of 18

18 10. Appendices Page 18 of 18