2013 ·Team Ciencias UNAM
WET LAB NOTEBOOK The beginnings Mid December 2012 After Pablo Padilla, our Synthetic Biology professor told us about iGEM and propose the idea of creating an iGEM team we had our first meeting, in which we discussed about ideas for this year project, from game theory to stemcells, we decided to start elaborating a project which consisted of recreating a predator-prey system in which the roles of the prey and the predator could interchange depending of the environmental conditions. This was our main purpose until we figured out how difficult it was to characterize this system and the technology we needed to develop a project like this.
Early June 2013 After deciding the best idea was to wait for next-years iGEM to present our prey-predator project, and that we needed a new project for this year, we started by analyzing the main goals of the iGEM competition. We needed a project in which we had to have two main BioBrick parts, one new and one from another year that we could improve, easy to characterize and validate, still if the overall project didn’t worked as expected. Given iGEM is a competition which looks for science improvement via synthetic biology, we though it was a good idea to think in a project that could have a direct implication in society, so it could benefit from this competition. With this in mind, we searched for a problem that could be solved with synthetic biology, so while having a meal at a suspiciously unhealthy fonda (dinner) in Mexico City we thought it would be a good idea if we designed a nonpathogenic bacteria that produced an antibiotic only in the presence of a pathogenic one. After reading an incredible amount of information about antimicrobial peptides and quorum-sensing molecules in pathogenic bacteria, we decided it was a good idea to focus on Helicobacter pylori health issue, a particularly problematic one in our country. Basically we needed an antimicrobial peptide that could inhibit H. pylori growth, and a quorum-sensing molecule that E. coli could sense, in order for it to produce the peptide. With this in mind we looked through past years iGEM projects. We took ideas for the quorum-sensing system from the team Calgary 2008, a great inspiration, given they had a similar idea in which bacteria they designed could be sensed and killed by another bacteria they designed. We wanted to kill a human natural pathogen, so the proteins they produced to kill the synthetic bacteria were not useful for us, so we came in touch with team Trieste 2012. They used LL-37 for theirs project, an antimicrobial peptide that we could use to inhibit H. pylori growth, although they could not produced the peptide, we though it was a good idea to try to improve theirs part. Finally we read the team NTU-Singapore 2008 wiki, they characterized a promoter that was activated with AI-2, a quorum-sensing molecule produced by some pathogenic bacteria (H. pylori among them).
SUMMER July 2013 JULY 3 RD 2013 : After designing our constructions, we ordered three sequences from Genscript; LL-37, the antimicrobial peptide used by team Trieste 2008, but with a different sequence; MarA, a multiple antibiotic resistance inductor in E.coli that has been proved to give resistance to LL-37 and other antibiotics. JULY 9 TH 2013: We started transforming our E. coli Top 10 with the iGEM parts we needed. Distribution
Plate
Plasmid Backbone LsrA promoter (indirectly activated by AI-2) 2013 Kit Plate 2 pSB1C3 Promoter (lacI regulated) 2013 Kit Plate 3 pSB1C3 IPTG inducible promoter with RBS 2013 Kit Plate 3 pSB1C3 RBS (Elowitz 1999) 2013 Kit Plate 5 pSB1A2 Green Fluorescent Protein 2013 Kit Plate 5 pSB1A2 LL 37 - Cathelicidin 2013 Kit Plate 1 pSB1C3 Terminator (simple) 2013 Kit Plate 3 pSB1C3 Double terminator (B0010-B0012) 2013 Kit Plate 3 pSB1C3 Lysis gene 2013 Kit Plate 3 pSB1C3 PBad promoter 2013 Kit Plate 3 pSB1C3 GFP Generator 2013 Kit Plate 3 pSB1C3
Well BBa_K117002 17C BBa_R0010 3H BBa_J04500 20J BBa_B0034 2M BBa_E0040 14K BBa_K875009 11N BBa_B1006 6D BBa_B0015 4F BBa_K117000 17H BBa_I13453 20O BBa_E0840 23C
We used the transformation protocol #1 to transform each of these parts into Top10 cells made competent by the protocol for competent cells #1. Given the vacation period we were having trouble finding a lab where we could work, so we did minipreps of all the colonies following the Maestrogen High-Speed Plasmid Mini Kit protocol to confirm the plasmids latter. COMPETENT CELLS PROTOCOL 1 Rubidium competent cells Protocol: 1. Grow a 5ml Overnight culture of the desired strain. 2. Add 1 ml of the Overnight culture to 2 separate flasks with 100 ml LB each and grow with shaking at 37째C to Optical Density 600 ~ 0.6. (Takes about 2 hours.) 3. Transfer the culture to two 50 ml conicals and chill on ice for 20 min. 4. Spin down the cells at 4째C max (5100RPM) in the Allegra 25R centrifuge for 5 min. 5. Pour off the supernatant and resuspend each pellet in 15 ml of TFBI(gently by hand) and shake on ice for 20 min. (taped to spinner in 4째C cold room). 6. Spin asin step 4 and discard the supernatant. 7. Gently resuspend each pellet in 2 mls of TFBII and incubate on ice for 30 minutes. 8. Aliquot the cells in 200 ul aliquots and freeze at-70째C in autoclaved labeled eppendorfs. *Try to do steps 3-8 in the cold room as much as possible. TFB I (30mM KOAc, 50mM MnCl2, 100mM RbCl, 10mM CaCl2, 15% (Glycerol) 250mL KoAc 1.472g 0.736 MnC12 4.95 g 2.475
RbC1 6.05 g 3.03 CaC12 (Dihydrate) 0.735 g 0.368 Glycerol 75 ml 37.5 Adjust pH to 5.8 with acetic acid. Did not need much dilute 1:10, few drops. Up to 500 ml with dH2O. Sterile filter and store at 4°C. TFB II (10mM NaMOPS, pH 7.0, 75mM CaC12, 10mM RbC1, 15% Glycerol) 250mL MOPS 1.046 g 0.523 CaC12·2H2O 5.513 g 2.757 RbC1 0.605 g 0.303 Glycerol 75 ml 37.5 Adjust pH to 7.0. Up to 500 ml with dH2O. Sterile filter and store at 4°C
Maestrogen High-Speed Plasmid Mini Kit protocol Step 1 Harvesting -Transfer 1.5 ml of cultured bacterial cells to a microcentrifuge tube. -Centrifuge at 14-16,000 x g for 1 minute and discard the supernatant. -If more than 1.5 ml of cultured bacterial cells is used, repeat the Harvesting Step. Step 2 Re-suspension -Add 200 µl of PD1 Buffer (RNase A added) to the tube and re-suspend the cell pellet by vortex or pipetting. Step 3 Lysis -Add 200 µl of PD2 Buffer and mix gently by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. -Let stand at room temperature for at least 2 minutes to ensure the lysate is homologous. Step 4 Neutralization -Add 300 µl of PD3 Buffer and mix immediately by inverting the tube 10 times. Do not vortex. -Centrifuge at 14-16,000 x g for 3 minutes. Step 5 DNA Binding -Place a PD Column in a 2 ml Collection Tube. -Add the supernatant from Step 4 to the PD Column and centrifuge at 14-16,000 x g for 30 seconds. -Discard the flow-through and place the PD Column back in the 2 ml Collection Tube. Step 6 Wash -Add 400 µl of W1 Buffer into the PD Column. -Centrifuge at 14-16,000 x g for 30 seconds. -Discard the flow-through and place the PD Column back in the 2 ml Collection Tube. -Add 600 µl of Wash Buffer (ethanol added) into the PD Column. -Centrifuge at 14-16,000 x g for 30 seconds. -Discard the flow through and place the PD Column back in the 2 ml Collection Tube. -Centrifuge at 14-16,000 x g again for 3 minutes to dry the column matrix Step 7 DNA Elution -Transfer the dried PD Column to a new microcentrifuge tube. -Add 50 µl of Elution Buffer or TE into the center of the column matrix. -Let stand for at least 2 minutes to allow the Elution Buffer or TE to be completely absorbed. -Centrifuge at 14-16,000 x g for 2 minutes to elute the DNA.
August 2013 AUGUST 4 TH 2013: Today our sequences from Genscript arrived, so we proceeded to obtain the lyophilized plasmid DNA with the protocol to obtain lyophilized DNA and transformed them with the transformation protocol #2 Transformation protocol #2 • Add 200 ng of supercoiled DNA to 40 µl of competent cells mixing gently. • Place in ice for one hour. • Heat at 42°C for one minute. • Places tubes in ice for five minutes. • Add 500 µl of SOC. • Incubate at 37°C with shaking at 250 rpm for one hour. • Plate the transformation mix onto the specific antibiotic plates. • Incubate at 37°C overnight.
Protocol to obtain lyophilized DNA. • Keep the vial sealed until ready to use. • Centrifuge at 6,000 x g for 1 minute at 4°C. Open the vial and add 20 µl of sterilized water to dissolve the DNA. • Close the lid and vortex the vial for 1 minute. If necessary, heat at 50°C for 15 minutes to dissolve the DNA. • Transformation of the plasmid DNA can be performed directly after the steps above. Verify sequence after each subcloning and transformation step.
AUGUST 6 TH 2013: In order to confirm our pending transformations from the iGEM parts we did the plasmid PCR protocol #1. Plasmid PCR protocol #1 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 1 µl DNA • 10 µl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X 100) • 5 µl 25 mM MgCl2 • 2 µl forward BioBrick primer • 2 µl reverse BioBrick primer • 2 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 1 µl Taq polymerase • 87 µl Water • 100 µl PCR conditions • 10 minutes at 95°C 1 cycle • 30 seconds at 95°C • 30 seconds at 55°C 25 cycles • 1 minute at 72°C • 5 minutes at 72°C 1 cycle
AUGUST 7 TH 2013: We did a 1% agarose gel electrophoresis with the PCR products from August 6th, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left: cl lambda (control), cl lambda, ladder, LacI, Lysis gene, LL-37 from Trieste, pBad, Terminator, GFP generator.
We discarded pBad and transformed two new parts from the registry with transformation protocol #2 into Top10 cells made competent by the protocol for competent cells #1. araC-Pbad 2013 Kit Plate 2 pLsra+YFP+term inator 2013 Kit Plate 3
pSB1C3 pSB1C3
BBa_K808000 7E BBa_K117008 17L
AUGUST 8 TH : In order to confirm our colonies we did the colony PCR protocol #1. Colony PCR protocol #1. Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 25 µl PCR Master Mix (Fermentas) • 1 µl forward BioBrick primer • 1 µl reverse BioBrick primer • 23 µl Water • 50 µl To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions • 10 minutes at 95°C 1 cycle • 30 seconds at 95°C • 30 seconds at 55°C 25 cycles • 50 seconds at 72°C • 5 minutes at 72°C 1 cycle
We did a 1% agarose gel electrophoresis with the PCR products, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left: cl lambda (control), LacI, Lysis Gene, LL-37 Trieste, ladder, LacI+RBS, terminator, GFP generator.
->Because we had very few colonies from the August 7th transformation, we grew them in a new plate. AUGUST 9 TH : In order to confirm the August 7th transformations, we did the colony PCR protocol #2. We did PCR to three colonies from each plate Colony PCR protocol #2 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 30 µl PCR Master Mix (Fermentas) • 1.2 µl forward BioBrick primer • 1.2 µl reverse BioBrick primer • 27.6 µl Water • 60 µl To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions • 10 minutes at 95°C 1 cycle • 30 seconds at 95°C • 30 seconds at 55°C 25 cycles • 50 seconds at 72°C 5 minutes at 72°C 1 cycle
We did a 1% agarose gel electrophoresis with the products of the PCR products from August 6th, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left: pLsra+YFP+terminator1, pLsra+YFP+terminator2, pLsra+YFP+terminator3, araC-Pbad1, araC-Pbad2, araC-Pbad3, ladder.
AUGUST 12 TH : We digested the following GFP generator with digestion protocol #1 by triplicate. pLsrA plasmid with digestion protocol #2. pLsrA+rbs+YFP+term plasmid with digestion protocol #3 by duplicate. LL-37 Trieste plasmid with digestion protocol #4. GFP generator with digestion protocol #3 by triplicate. RBS plasmid with digestion protocol #5. araC+pBad plasmid with digestion protocol #1 by duplicate. We did the colony PCR protocol #3 to confirm the Genscript transformations from August 4th. Digestion protocol #1 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 1 µl DNA • 2.5 µl R buffer • 0.5 µl EcoRI • 2.0 µl SpeI • 19 µl Water • 25 µl Incubate at 37°C overnight.
Digestion protocol #2 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 1 µl DNA • 5 µl Cutsmart 2X buffer • 0.5 µl EcoRI • 1.0 µl XbaI • 17.5 µl Water • 25 µl Incubate at 37°C overnight.
Digestion protocol #3 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 1 µl DNA • 2.5 µl Cutsmart buffer • 0.5 µl XbaI • 0.5 µl PstI • 21.5 µl Water • 25 µl Incubate at 37°C overnight.
Digestion protocol #4 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 1 µl DNA • 2.5 µl Cutsmart buffer • 0.5 µl SpeI • 0.5 µl PstI • 21.5 µl Water • 25 µl Incubate at 37°C overnight.
Digestion protocol #5 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 0.5 µl DNA • 5 µl Cutsmart 2X buffer • 0.5 µl EcoRI • 1.0 µl XbaI • 18 µl Water • 25 µl Incubate at 37°C overnight.
AUGUST 13 TH : We did a gel electrophoresis to confirm the digestions from August 12th before purifying, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left: pLacI-RBS (s/p), MarA (x/p), MarA (x/p), MarA (x/p), RBS-GFP-term (e/s), RBS-GFP-term (e/s), ladder, RBS-GFP-term (e/s), pLsrA (e/x), simpleterminator (s/p), pLsrA-rbs-YFP-term (x/p), pLsrA-rbs-YFP-term (x/p), LL-37 (s/p), LL-37 from Trieste (s/p), RBS-GFP-term (x/p), RBS-GFP-term (x/p), ladder, RBS-GFP-term (x/p), RBS (e/x), araC-pBad (e/s), araC-pBad (e/s). These are the gels, from which we purified.
From left: pSB1C3, ladder.
From left: ladder, MarA (x/p), ladder, RBS-GFP-term (e/s).
From left: pLsrA-rbs-YFP-term (x/p), ladder, RBS-GFP-term (x/p), ladder, araC-pBad (e/s).
After purifying the vectors from gel electrophoresis with protocol X, and the inserts with protocol Y we ligated the digested fragments with ligation protocol #1. Ligation protocol #1. Calculate the volume of the vector and the insert to have a molar relation 3:1 of vector:intert, using 100 ng of vector with the following data: !"#$%&!!"!!"#$%&!(!") ! = !
!"#$%!!"#$%&!(!")!×!!"#$!!"!!"#$%&!(!") !"#$%!!"#$%!!"!!ℎ!!!"#$%& ×! !"#$!!"!!"#$%&!(!") !"#$%!!"#$%!!"!!ℎ!!!"#$%&
We ligated in the following order: 1) BBa_K1230008.-pLsrA (e/x) with rbsGFPterm (e/s) 2) BBa_K1230002.-term (s/p) with pLsrA-YFP-term (x/p) 3) BBa_K1230003.-LL-37 from Trieste (s/p) with rbsGFPterm (x/p) 4) BBa_K1230009.-RBS (e/x) with araCpBad (e/s) Given the small size of LL-37 plasmid we ordered from Genscript, we had to amplify it with PCR to insert them in pSB1C3. We used the plasmid PCR protocol #2. Plasmid PCR protocol #2. Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 0.5 µl DNA • 5 µl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X 100) • 3 µl 25 mM MgCl2 • 1 µl forward pUC57 primer • 1 µl reverse pUC57 primer • 1 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 0.5 µl Taq polymerase from Vivantis. • 38 µl Water • 50 µl PCR conditions • 3 minutes at 95°C 1 cycle • 30 seconds at 95°C • 30 seconds at 55°C 25 cycles • 1 minute at 72°C • 5 minutes at 72°C 1 cycle
AUGUST 14 TH : Because plasmid PCR protocol #2 didn’t go as expected, we are amplifying again LL-37 with plasmid PCR protocol #3. Plasmid PCR protocol #3. Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 0.2 µl DNA • 5 µl Buffer A • 3 µl 25 mM MgCl2 • 1 µl forward pUC57 primer • 1 µl reverse pUC57 primer • 1 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 0.3 µl Taq polymerase from Vivantis. • 38.7 µl Water • 50 µl
PCR conditions • 3 minutes at 94°C • 30 seconds at 94°C • 30 seconds at 55°C • 25 minute at 72°C • 5 minutes at 72°C
1 cycle 25 cycles 1 cycle
After amplifying the plasmid, we digested LL-37 in order to put it into pSB1C3 with EcoRI and SpeI with Digestion protocol #1. We transformed all the ligations from August 13th with transformation protocol #2. AUGUST 16 TH : We did a gel electrophoresis to confirm the digestions from August 15th before purifying, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left:, pSB1C3 (e/s), LL-37 (e/s), ladder, LL-37 e/s)
We ligated LL-37 in order to put it into pSB1C3 with ligation protocol #1. In order to confirm the August 14th transformations we did the colony PCR protocol #3. We also did a PCR to confirm MarA and LL-37 with the colony PCR protocol #5. We did PCR to five colonies from each plate. Colony PCR protocol #3. Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 5 µl Buffer A • 3 µl 25 mM MgCl2 • 1 µl forward BioBrick primer • 1 µl reverse BioBrick primer • 1 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 0.3 µl Taq polymerase from Vivantis. • 38.7 µl Water • 50 µl To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions • 6 minutes at 94°C 1 cycle • 30 seconds at 94°C • 30 seconds at 55°C 25 cycles • 40 seconds at 72°C • 5 minutes at 72°C 1 cycle
Colony PCR protocol #5. Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 5 µl Buffer A • 3 µl 25 mM MgCl2 • 1 µl forward pUC57 primer • 1 µl reverse pUC57 primer • 1 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 0.3 µl Taq polymerase from Vivantis. • 38.7 µl Water • 50 µl To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions • 6 minutes at 94°C 1 cycle • 30 seconds at 94°C • 30 seconds at 55°C 25 cycles • 40 seconds at 72°C • 5 minutes at 72°C 1 cycle
We did a 1% agarose gel electrophoresis to see this PCR, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left:, BBa_K1230008, BBa_K1230008, BBa_K1230008, BBa_K1230008, BBa_K1230002, BBa_K1230002, BBa_K1230002, BBa_K1230002, BBa_K1230002, , ladder, ladder, MarA, MarA, LL37, BBa_K1230003, BBa_K1230003, BBa_K1230003, BBa_K1230003, BBa_K1230003, BBa_K1230009, BBa_K1230009, BBa_K1230009, BBa_K1230009, BBa_K1230009.
AUGUST 17 TH : We did a the colony PCR protocol #3 to confirm MarA inside pSB1C3. We did PCR to five colonies from each plate. We did a 1% agarose gel electrophoresis to see this PCR, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left:, MarA, MarA, MarA, MarA, ladder, ladder, MarA, MarA, MarA, MarA
AUGUST 20 TH : We did a the colony PCR protocol #3 to confirm LL37 inside pSB1C3. We did a 1% agarose gel electrophoresis to see this PCR, using the 1 kb plus ladder from Invitrogen (positive, negative)
From left:, LL-37, ladder
Back to school SEPTEMBER 2013 SEPTEMBER 1 ST : We digested: BBa_K1230008 with digestion protocol #8. MarA plasmid with digestion protocol #9. rbs+GFP+term plasmid with digestion protocol #8. LacI+rbs plasmid with digestion protocol #10. LacI+rbs with digestion protocol #11. LL-37 plasmid with digestion protocol #10. araC+pBad plasmid with digestion protocol #1 by duplicate. BBa_K1230009 with digestion protocol #8. Digestion protocol #8 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 10 µl DNA • 0.5 µl Cutsmart buffer • 1 µl EcoRI • 2 µl SpeI • 6.5 µl Water • 20 µl Incubate at 37°C for 8 hours Digestion protocol #9 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 10 µl DNA • 2 µl 2.1 buffer • 1 µl XbaI • 1.5 µl PstI • 5.5 µl Water • 20 µl Incubate at 37°C for 8 hours Digestion protocol #10 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 10 µl DNA • 2 µl Cutsmart buffer. • 1 µl XbaI • 1. µl EcoRI • 6 µl Water • 20 µl Incubate at 37°C for 8 hours
Digestion protocol #11 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 10 µl DNA • 2 µl Cutsmart buffer. • 1 µl SpeI • 1. µl PstI • 6 µl Water • 20 µl Incubate at 37°C for 8 hours
SEPTEMBER 2 ND : We did a 1% agarose gel electrophoresis to see this PCR, using the AccuRuler100 kb plus ladder from MaestroGen (positive, negative)
From left:, MarA (x/p), LacI (e/x), LL-37 (e/x), ladder, BBa_K1230009 (e/s), BBa_K1230008 (e/s), LacI (s/p), GFP generator (e/s).
We purified the fragments from the gel electrophoresis using the Silica Bead DNA Gel Extraction Kit from ThermoScientific. Silica Bead DNA Gel Extraction Kit from ThermoScientific. 1 Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice. Note 2 If the purified DNA will be used for cloning, avoid UV damage of the DNA by minimizing the UV exposure to a few seconds or keeping the gel slice on a glass or plastic plate during UV illumination. Add a 3:1 volume of Binding Buffer to the gel slice (volume:weight) (e.g., add 300 µl of Binding Buffer for every 100 mg of agarose gel). Incubate the gel mixture at 55°C for 5 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Note If the DNA is extracted from a TBE agarose gel, add 1⁄2 volume of TBE Conversion Buffer and 4.5 volumes of Binding Buffer to a given volume of agarose. 3 Add the resuspended Silica Powder Suspension to the DNA/Binding Buffer mixture. For ≤2.5 µg of DNA add 5 µl of Silica Powder Suspension. For >2.5 µg of DNA add 2 µl of Silica Powder Suspension per µg of DNA. Incubate the mixture for 5 min at 55°C to allow for binding of the DNA to the silica matrix. Mix by vortexing every few minutes to keep the silica powder in suspension. Note If a large amount of DNA is purified or if the volume of the binding reaction is greater than 1.5 ml increase the incubation time of the binding step to 15 min. 4 Spin the silica powder/DNA mixture for 5 s to form a pellet. Carefully remove the supernatant solution and discard. (Optional). The pelleted silica powder/DNA complex can be resuspended with an additional 300 µl of Binding
Buffer to dissolve any residual undissolved agarose from step 2. Place the suspension in a 55°C water bath for a few minutes and proceed to step 5. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow. 5 Add 500 µl of ice cold Washing Buffer (diluted with ethanol as described on p. 3), resuspend the pellet and spin for 5 s. Discard the supernatant. Repeat this procedure three times. After the supernatant from the last wash has been removed, spin the tube again and remove the remaining liquid with a pipette. If necessary air-dry the pellet for 10-15 min to avoid the presence residual ethanol in the purified DNA solution. Residual of ethanol in the DNA sample may inhibit downstream enzymatic reactions. Note • To obtain efficient washing, the pellet should be resuspended completely. Resuspend the pellet in the Washing Buffer by vortexing, pipetting the solution back and forth onto the pellet or manually resuspending the pellet with a pipette tip. • ForDNAfragments≥5kb, resuspend the pelletbyinvertingthetube.Vortexing can cause shearing of large DNA molecules 6 Resuspend the pellet in the desired volume of sterile deionized water or TE and incubate the tube at 55°C for 5 min. Spin the tube and remove the supernatant while avoiding the pellet. Place the recovered supernatant into a fresh tube and repeat the elution with another aliquot 6 of water or TE. For the removal of residual silica powder, spin the tube again for 30 s in a table-top centrifuge and transfer the supernatant into a new tube. Note. The optimal elution volume is equal to the volume of the silica powder suspension that was added in step 3.
After purifying the fragments we ligated them using the ligation protocol #1. BBa_K1230011.- BBa_K1230009+LL37 BBa_K1230012.- BBa_K1230008+LL37 BBa_K1230004.-LacIrbs+MarA Failed.-GFP+LacIrbs SEPTEMBER 3 RD : We transformed the ligations from September 2nd using the transformation protocol #3 using the competent cells prepared with the competent cells protocol #2. Transformation protocol #3 • Unfreeze the cells on ice. • Add 200 ng of supercoiled DNA, or up to 5 µl of ligation to 40 µl of competent cells mixing gently. • Place in ice for 10 to 30 minutes. • Heat at 42°C for one minute and a half. • Incubate on ice for 2 minutes. • Resuspend the cells on 1 ml of LB and incubate at 37°C with shaking at 250 rpm for 40 minutes. • Plate the transformation mix onto the specific antibiotic plates. • Incubate at 37°C overnight.
6 Given our characterization requirements, we transformed BBa_K1230004 on dh5α, BBa_K1230011 on Top10 and BBa_K1230012 on a LuxS- strain. SEPTEMBER 5 TH : In order to confirm the September 3rd transformations we did the colony PCR protocol #6. We did PCR to five colonies from each plate. We did a 1% agarose gel electrophoresis to see this PCR, using the AccuRuler100 kb plus ladder from MaestroGen (positive, negative) Colony PCR protocol #6 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). • 5 µl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X 100) • 3 µl 25 mM MgCl2 • 1 µl forward pUC57 primer • 1 µl reverse pUC57 primer • 1 µl 10mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) • 0.5 µl Taq polymerase.
• 38 µl Water • 50 µl To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions • 5 minutes at 95°C 1 cycle • 30 seconds at 94°C • 30 seconds at 55°C 25 cycles • 90 seconds at 72°C • 5 minutes at 72°C 1 cycle
From left: Control, BBa_K1230008, BBa_K1230009, BBa_K1230010, ladder, BBa_K1230004, BBa_K1230004, BBa_K1230004, BBa_K1230004, BBa_K1230004, ladder,
From left: BBa_K1230012, BBa_K1230012, BBa_K1230011, BBa_K1230011.
BBa_K1230012,
ladder,
BBa_K1230011,
BBa_K1230011,
BBa_K1230011,
SEPTEMBER 8 TH : In order to be sure everything is going on as expected until now with our constructions, we confirmed them by digestion, using the digestion protocol #12 Digestion protocol #12 Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot. • 10 µl DNA • 2 µl Cutsmart buffer. • 1 µl PstI • 1. µl EcoRI • 6 µl Water • 20 µl Incubate at 37°C for 8 hours
SEPTEMBER 9 TH : We did a 1% agarose gel electrophoresis to see the digestions from September 8th, using the AccuRuler100 kb plus ladder from MaestroGen (positive, negative)
From left: ladder, BBa_K1230008, BBa_K1230008 (e/p), BBa_K1230002, BBa_K1230008 (e/p), BBa_K1230003, BBa_K1230003 (e/p), ladder, BBa_K1230009, BBa_K1230009 (e/p), GFP generator, GFP generator (e/p), LacI-rbs, LacI-rbs (e/p), ladder, LL37, LL-37 (e/p), MarA, MarA (e/p), LL-37 from Trieste, LL-37 from Trieste (e/p), YFP generator (e/p), ladder, YFP generator, BBa_K1230004 (e/p), BBa_K1230004.
SEPTEMBER 16 TH : As we confirmed the parts we needed for our characterization, we started with our characterization. For this we inoculated in 3 ml of LB at 37°C overnight our Multiple Antibiotic Resistance generator part (BBa_K1230004) and the strain it was transformed into, but without the plasmid. Here we made a great mistake, the strain it was transformed into was dh5α, so we used the first dh5α strain we found on the fridge, but it was not from the same plate we made competent cells. Of course this results cannot be compared, so this experiment is not part of our characterization process. Nevertheless it was our first characterization experiment, and part of the development of our project, so we are going to present our results. We did the experiment in ten plates, 5 of them had LB-agar and 25 µg/ml of Chloramphenicol for selection pressure for our plasmid, and 5 of them just LB-agar for our control. In each group of five, each plate had a different IPTG concentration, 1mM, 0.75 mM, 0.50 mM, 0.25 mM and 0mM. In order to improve the LL-37 antimicrobial peptide part from Trieste, which was under the induction of IPTG, so we changed it to the arabinose promoter, this is because LacI has a really high basal level, and in a high copy vector, there is a great selection against having the plasmid, which can be one of the reasons this team had many problems characterizing it. pBad promoter has a basal level of almost 0, so we think inducing it with arabinose could make a characterization with more conclusive results.
To do this we made a preinoculus with the cells that had the LL-37 generator and the strain without the plasmid we used to transformed this device.
SEPTEMBER 17 TH : We used inhibition disks with different concentrations of tetracycline 1.25 µl/ml. 9 disks per plate. We did them like this. 0.2 µg/ml 0.5 µg/ml
1 µg/ml
50 µg/ml
0 µg/ml
2 µg/ml
20 µg/ml 10 µg/ml
5 µg/ml
We let them grow over-night at 37°C. This table show our results. Tetracycline concentration ug/ml (MarA overexpression) 0 0.2 0.5 1 2 5 10 20 50 0 0.2 0.5 1 2 5 10 20 50 0 0.2 0.5 1 2 5 10 20 50 0 0.2 0.5 1 2 5 10 20 50 0 0.2 0.5 1 2 5 10 20 50 0 0.2 0.5 1 2 5 10 20 50
Inhibition diameter mm 8 8 8 8 8 8 8.5 9 10 8 8.5 9 14 18 21 24 26 28 8 13 13 14 17 19 25 30 32 8 11 10 13 20 25 26 28 30 8 12 13 15 16 20 21 25 30 8 10 13 15 17 19 28 34 40
IPTG concentration mM LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG LB without Chloramphenicol nor IPTG 0 0 0 0 0 0 0 0 0 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 1 1 1 1 1 1 1 1 1
SEPTEMBER 18 TH : For the characterization of the LL-37 antimicrobial peptide from Trieste, with another promoter, we inoculated 12.5 ml of LB of the strain without the plasmid, and the strain with the plasmid (we made pressure selection with chloramphenicol to make sure our plasmid was still there). We did the inoculation at 0.05 of OD at 600A and incubating at 37째C until it got to 4.0 OD at 600A. Then we separated each of the two tubes in 5 15ml falcon tubes, 2 ml of the inoculums. We induced with arabinose as we mention in the following table. From each tube we did 8 decimal dilutions in 1.5 Eppendorf tubes with LB. These are the results. Control Top10
10-1
10-2
10-3
10-4 10-5 10-6 10-7
0 0.25 0.50
10-1 10-1 10-1
0.75 1 LL37 ara
0
10-1 10-2
0.25 0.50
10-1 10-1
0.75
10-1
10-3 10-4
10-5
10-6
10-7
1
SEPTEMBER 19 TH : We inoculated in 3 ml of LB at 37째C overnight our Multiple Antibiotic Resistance generator part (BBa_K1230004) and the strain it was transformed into, but without the plasmid. This time we used the same strain we transformed into.
SEPTEMBER 23 TH : For our final charachterization experiment for the MarA generator, we inoculated in 3 ml of LB at 37째C overnight our Multiple Antibiotic Resistance generator part (BBa_K1230004) and the strain it was transformed into, but without the plasmid, using the same strain we transformed into. For this we made 10 chloramphenicol plates, using another ten without chloramphenicol for the strain without the plasmid: 10->LB+IPTG (Different IPTG concentrations) 10->LB+IPTG+Chloramphenicol (Different IPTG concentrations) The IPTG concentrations are 0mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM.
SEPTEMBER 24 TH : This is how we prepared the different inhibition disks. 0 µg/ml
2 µg/ml
1 µg/ml
0.5 µg/ml
20 µg/ml
10 µg/ml
5 µg/ml
SEPTEMBER 25 TH : This is our final characterization experiment. We measured the Kanamycin resistance our construction gave to E. coli. For this, we used again the inhibition disks, with the different concentrations we mention in the following table. Control disks
µg of stock 1:10 Kanamycin 25 mg/ml
Inhibition diameter
IPTG concentration
MarA overproduction
µg of stock 1:10 Kanamycin 25 mg/ml
Inhibition diameter
IPTG concentration
0 µg/ml
0 ml
8 mm
0 mM
0 µg/ml
0 ml
8 mm
0 mM
0 mM
0.5 µg/ml
0.2 ml
10 mm
0 mM
0.4 ml
8 mm
0 mM
0.5 µg/ml
0.2 ml
15 mm
1.0 µg/ml
0.4 ml
11 mm
0 mM
1.0 µg/ml
2.0 µg/ml
0.8 ml
13 mm
0 mM
2.0 µg/ml
0.8 ml
10 mm
0 mM
5.0 µg/ml
2.0 ml
15 mm
0 mM
5.0 µg/ml
2.0 ml
13 mm
0 mM
10 µg/ml
4.0 ml
20 mm
0 mM
10 µg/ml
4.0 ml
18 mm
0 mM
20 µg/ml
8.0 ml
26 mm
0 mM
20 µg/ml
8.0 ml
19 mm
0 mM
0 µg/ml
0 ml
8 mm
0.25 mM
0 µg/ml
0 ml
8 mm
0.25 mM
0.5 µg/ml
0.2 ml
13 mm
0.25 mM
0.5 µg/ml
0.2 ml
10 mm
0.25 mM
1.0 µg/ml
0.4 ml
9 mm
0.25 mM
1.0 µg/ml
0.4 ml
9 mm
0.25 mM
2.0 µg/ml
0.8 ml
11 mm
0.25 mM
2.0 µg/ml
0.8 ml
9.5 mm
0.25 mM
0.25 mM
5.0 µg/ml
2.0 ml
12 mm
0.25 mM
0.25 mM
10 µg/ml
4.0 ml
13 mm
0.25 mM
8.0 ml
15 mm
0.25 mM
5.0 µg/ml 10 µg/ml
2.0 ml 4.0 ml
17 mm 20 mm
20 µg/ml
8.0 ml
26 mm
0.25 mM
20 µg/ml
0 µg/ml
0 ml
8 mm
0.5 mM
0 µg/ml
0 ml
8 mm
0.5 mM
0.5 µg/ml
0.2 ml
9 mm
0.5 mM
0.5 µg/ml
0.2 ml
11 mm
0.5 mM
1.0 µg/ml
0.4 ml
10 mm
0.5 mM
1.0 µg/ml
0.4 ml
9 mm
0.5 mM
2.0 µg/ml
0.8 ml
12 mm
0.5 mM
2.0 µg/ml
0.8 ml
10 mm
0.5 mM
5.0 µg/ml
2.0 ml
16 mm
0.5 mM
5.0 µg/ml
2.0 ml
12 mm
0.5 mM
10 µg/ml
4.0 ml
21 mm
0.5 mM
10 µg/ml
4.0 ml
15 mm
0.5 mM
20 µg/ml
8.0 ml
26 mm
0.5 mM
20 µg/ml
8.0 ml
19 mm
0.5 mM
0 µg/ml
0 ml
8 mm
0.75 mM
0 µg/ml
0 ml
8 mm
0.75 mM
0.75 mM
0.5 µg/ml
0.2 ml
10 mm
0.75 mM
0.75 mM
1.0 µg/ml
0.4 ml
8.5 mm
0.75 mM
0.8 ml
9 mm
0.75 mM
0.5 µg/ml 1.0 µg/ml
0.2 ml 0.4 ml
19 mm 9 mm
2.0 µg/ml
0.8 ml
12 mm
0.75 mM
2.0 µg/ml
5.0 µg/ml
2.0 ml
17 mm
0.75 mM
5.0 µg/ml
2.0 ml
11 mm
0.75 mM
10 µg/ml
4.0 ml
19 mm
0.75 mM
10 µg/ml
4.0 ml
14 mm
0.75 mM
20 µg/ml
8.0 ml
24 mm
0.75 mM
20 µg/ml
8.0 ml
16 mm
0.75 mM
0 µg/ml
0 ml
8 mm
1 mM
0 µg/ml
0 ml
8 mm
1 mM
0.5 µg/ml
0.2 ml
18 mm
1 mM
0.5 µg/ml
0.2 ml
10 mm
1 mM
1.0 µg/ml
0.4 ml
10 mm
1 mM
1.0 µg/ml
0.4 ml
9 mm
1 mM
2.0 µg/ml
0.8 ml
11 mm
1 mM
2.0 µg/ml
0.8 ml
9.5 mm
1 mM
5.0 µg/ml
2.0 ml
17 mm
1 mM
5.0 µg/ml
2.0 ml
11 mm
1 mM
10 µg/ml
4.0 ml
21 mm
1 mM
10 µg/ml
4.0 ml
15 mm
1 mM
20 µg/ml
8.0 ml
19 mm
1 mM
20 µg/ml
8.0 ml
29 mm
1 mM
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