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Notebook

1.06.15

1. Preparation of the LB agar
   We used 37 g of nutrient agar for 400 mL of distilled water
2. Extraction of genome from S.mutans
   
   1. First, culture S.mutans in 5 mL liquid BHI + bacitracin
   2. Centrifuge for 15 min at 4000 rpm
   3. Then dissolve the pellet in 500 microliters of Lysis Buffer
      How to prepare Lysis Buffer (1 mL):
      Lysis Buffer contains EDTA, Tween 80%, tris HCl,125 microliters of 8 M EDTA,5 µl of Tween 80, Tris HCl 1M 50 µl, Proteinase K (200 µg/mL). 0.0002 grams of powder Proteinase K were put into 1 mL of Lysis Buffer. The balance could not read 0.0002 g of proteinase K, so 0.02 g of proteinase K were taken.
3. Incubation for 2 hours at 55°C. Heat at 90°C for 5 minutes.
4. Then add equal volume of cold isopropyl alcohol.
5. Incubate in freezer for 20 minutes.
6. Centrifuge at the maximum speed for 30 min. Remove the supernatant.
7. Add enough amount of ethanol to the pellet in order to wash
8. Then add 50 µl of TE buffer
9. Add 0.5 µl of the RNAase
10. Incubate at 37°C for 1 hour
11. Inactivate at 60°C for 10 min
12. Run it in an agarose gel



2.06.15

1. Construction of the light system
   pFixK2(K592006) + rbs + tetR(C0040) + double terminator + Ptet(R0040) + rbs + RFP(J06505) + double terminator
   • [rbs] = 139.15 ng/µl
   • [Ptet + GFP] = 199.2 ng/µl
   • [pFixK2] = 131.6 ng/µl
   • [double terminator] = 70.21 ng/µl
   • [tetR] = 78.76 ng/µl
Restriction digest of pFixK2 and RBS 1. BBa_K592006 FixK2 promoter is the 250 base pairs long. It was cut with NEB enzymes, EcoRI and SpeI. · pFixK2 – 7.6 µl · EcoRI = 0.5 µl · SpeI = 0.5 µl · dH2O = 36.4 µl · cut smart = 5 µl Overall: 50 µl 2. Ribosome binding site (15 base pairs) was cut with SpeI NEB Enzyme · RBS = 7.2 µl · XbaI = 1 µl · Cut smart = 5 µl · dH2O = 36.8 µl Overall: 50 µl Restriction digest of tetR and double terminator 1. tetR (685 base pairs) was cut with EcoRI and SpeI. · tetR = 12.7 µl · EcoRI= 0.5 µl · SpeI = 0.5 µl · Cut smart = 5 µl · dH2O = 31.3 µl Overall: 50 µl 2. Double terminator (95 base pairs ) · dTer = 14.2 µl · XbaI = 1 µl · Cut Smart = 5 µl · dH2O = 29.8 µl Overall: 50 µl Gel extraction of the pFixK2, RBS, tetR and double terminator 1. Invitrogen by life Technologies PureLink Quick Gel Extraction Kit was used to purify DNA. 2. The small area of the gel containing the DNA fragment of interest was cut under UV. 3. Mass of FixK2 = 220 mg RBS = 80 mg tetR = 150 mg dTer = 140 mg 4. The protocol of the PureLink Quick Gel Extraction was used to dissolve the gel and extract the DNA 5. [FixK2] = 5.727 ng/µl [Rbs] = 4.499 ng/µl [tetR] = 5.216 ng/µl [double terminator] = 2.694 ng/µl Ligation of the Parts pFixK2+ rbs · pFixK2 = 8.5 µl · RBS = 8.5 µl · T4 ligase = 1 µl · T4 buffer = 2 µl Overall: 20 µl tetR +double terminator · tetR = 8.5 µl · double terminator= 8.5 µl · T4 ligase = 1 µl · T4 buffer = 2 µl Overall: 20 µl Then ligated DNA was transformed and plate with pFixK2 with RBS grew on the plate. The mini- prep of the pFixK2 + rbs was done 3.06.15 The concentration of the transformed ligated parts FixK2+ rbs= 75.79 ng/µl FixK2+ rbs= = 72.80 ng/µl tetR+dter= 56. 93 ng/µl tetR+dter= 95.86ng/µl The concentration of the transformermed Pveg and FixJ Pveg (BBa_K823003) and FixJ (BBa_K592016) Pveg= 84.84 ng/µl FixJ= 161.1 ng/µl PCR (Thermo Scientific Phusion High Fidelity PCR Master- mix ) 1. FixK2 · DNA = 0.03 µl *10 reactions = 0.3 µl · Water = 5.97 µl= 59.7 µl · Master Mix = 10 µl= 100 µl · VF2 = 2µl = 20 µl · VR= 2 µl = 20 µl 2. RBS · DNA = 0.0277 µl = 0.287 µl · Water= 5.9713 µl = 59.7 µl · MM= 10 µl = 100 µl · VF2= 2 µl = 20 µl · VR=2 µl = 20 µl 3. FixK2+ rbs · DNA= 0.06 =0.6 · Water= 5.94=59.4 · MM=10= 100 · VF2=2=20 · VR= 2=20 4. tetR+dter · DNA= 0.07=0.7 · Water= 5.93=59.3 · MM= 10=100 · VF2= 2= 20 · VR=2 =20 5. tetR · DNA= 0.05= 0.5 · Water= 5.95= 59.5 · MM= 10 =100 · VF2= 2= 20 · VR= 2= 20 6. Double terminator · DNA= 0.06=0.6 · Water= 5.94= 59.4 · MM= 10 =100 · VF2=2= 20 · VR=2=20 Results; The ligation of the FixK2+ rbs did not work The ligation of the tetR+ dter worked The Restriction Digest of the tetR (BBa_C0040) and double terminator (BBa_K823017) tetR · Water= 31.3 µl · DNA= 12.7 · EcoR1= 0.5 · SpeI= 0.5 · Cut Smart= 5 Double terminator · Water= 29.8 µl · DNA= 14.2 · EcoRI= 0.5 · XbaI = 0.5 · Cut Smart= 5 µl Concentrations after Gel extraction 4.06.15 #1 PCR of the ligated parts after transformation and mini - prep [FixK2+rbs+tetR] (950 base pairs) = 108.54 ng/µl [tetR+ double terminator] = 166 ng/µl 1. FixK2+rbs+tetR · High Fidelity Master Mix= 10*10 reactions= 100 µl · DNA= 0.09=0.9 µl · VF2 (0.5 µM) = 2 µl = 20 µl · VR= 2 µl · Sterile water =5.91 µl 2. tetR + double terminator · High Fidelity Master Mix= 10µl = 100 µl · tetR+ double terminator= 0.06= 0.6 µl · VF2= 2 =20 µl · VR= 2=20 µl · Sterile water= 5.94= 59.4 µl #2 Running on the gel of the parts after PCR Order of the parts on the gel; 1. Ladder 2. FixK2+ RBS 3. FixK2+ rbs+tetR 4. tetR 5. tetR+ dter 5.06.15 Protocol for making the competent dh5alpha 1. Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap) 2. Grow on 37°C with shaking for 130 rpm overnight 3. Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning 4. Shake 37°C for 1.5-3 hours 5. When OD is between 0.3-0.4 put cell on ICE 6. Hold cells on ice for the 10 minutes 7. Collect cells by centrifugation for 3 min at maximum speed (4700 rpm) 8. Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2 (prepared in ddH2O). Treat them gently. 9. Incubate on ice for 20 minutes. 10. Centrifuge again at maximum speed (4700 rpm) 11. Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol) 12. Dispence into chilled microtubes, put on the dry ice. Perform this procedure very quickly! 13. Freeze in -80°C 6.06.15 #1 PCR clean-up of the [FixK2+rbs] = 30.15 ng/µl [tetR] = 57.47 ng/ #2 [FixK2 + rbs] (PCR product digested) = 5.862 ng/µl [tetR] = 4.621 ng/µl #3 Fermentas Ligation tetR and double terminator [tetR] = 20/4.621ng/µl = 4.3 µl [FixK2+ rbs] = 40ng/5.862 ng/µl = 7 µl Sterile water = 1µl Ligase= 1 µl Buffer = 2 µl #4 Ligation of · Pveg + FixJ · FixK+ rbs+tetR · tetR+double terminator Ligation of tetR with double terminator with NEB- enzymes 1:1 50 ng: 50 ng tetR = 7.518 ng/µl tetR; 6.7 ng/ µl double terminator; 3.3 µl T4 ligase: 1 µl T4 buffer: 2 µl Sterile water: 7 µl Pveg (19.59)+ FixJ (8.968)(Fermentas) 50ng: 50 ng Pveg =2 µl FixJ = 5.6 µl T4= 1 µl Buffer= 2 µl Sterile water= 8.8 µl FixK2+ rbs(10.66 ng/µl)+ tetR (23.08ng/µl) (NEB) FixK2+ rbs = 4.7 µl tetR= 2.2 µl T4-ligase = 1µl T4-buffer= 2 µl Sterile water = 10.1 µl tetR(4 ng/µl)+ double terminator (2ng/µl)(Fermentas) tetR= 8 µl double terminator= 9 µl T4 ligase = 1 µl Buffer= 2 µl tetR + double terminator (Fermentas ) 25 ng: 25 ng tetR= 5 µl double terminator=12 µl T4 ligase = 1 µl Buffer = 2 µl Results; Ligation of the tetR + double terminator (NEB) 50 ng: 50 ng have worked 8.06.15 #1 Transformation of the Ligated products of the · Pveg + FixJ · tetR+ double terminator · FixK2+rbs+tetR · FixK2+rbs+tetR (PCR) · FixK2+rbs+tetR · FixK2+tetR+ double terminator · tetR+ double terminator 9.06.15 #1 · Pveg +FixJ = 98.29 ng/µl · tetR+ double terminator = 65.34 ng/µl · FixK2+ rbs+ tetR = 83.32 ng/µl · FixK2+ rbs+ tetR = 81.64 ng/µl · tetR+ double terminator (NEB) = 101.3 ng/µl · tetR+ double terminator = 100.4 ng/µl #2 Polymerase chain Reaction 50µl 4 Master Mix 25µl 100 µl tetR+ double terminator 0.15 µl 0.6 µl VF2 5µl 20 µl VR 5µl 20µl H2O 14.8 µl 59.4 µl 50µl 4 Master Mix 25µl 100 µl FixK2+rbs+ tetR 0.12 µl 0.48 µl VF2 5µl 20µl VR 5µl 20µl H2O 14.88 µl 59.52 µl 50µl 4 Master Mix 25µl 100 µl Pveg (237 base) + FixJ (1796) 0.1 µl 0.4 µl VF2 5µl 20 µl VR 5µl 20µl H2O 14.9 µl 59.6 µl 50µl 4 Master Mix 25µl 100 µl FixK2+rbs+tetR 0.12 µl 0.48 µl VF2 5µl 20 µl VR 5µl 20µl H2O 14.88 µl 59.52µl 50µl 4 Master Mix 25µl 100 µl tetR+ double terminator (Fermentas) 0.1 µl 59.6 µl VF2 5µl 20 µl VR 5µl 20µl H2O 14.9 µl 59.4 µl 50µl 4 Master Mix 25µl 100 µl tetR+ double terminator (fermentas) (2:1 fermentas) 0.1 µl 0.4 µl VF2 5µl 20 µl VR 5µl 20µl H2O 14.9 µl 59.6 µl Results: Only the ligation of the tetR + double terminator with NEB enzyme have worked 10.06.15 #1 Restriction Digest of the FixK2+ rbs with EcoRI and SpeI DNA: 1000 ng/ 75.79ng/µl = 13.2 µl EcoRI: 0.5 µl SpeI: 0.5 µl Cut smart: 5 µl Sterile water: 30.8 µl #2 Restriction Digest of tetR + double terminator with EcoRI and XbaI DNA: 1000 ng/101.3 ng/µl = 9.87 µl EcorI: 0.5 µl XbaI: 0.5 µl Cut smart: 5 µl Sterile water: 34.13 µl 11.06.15 #1 Gel extract of the Pveg and FixJ [Pveg ] = 3.174 ng/µl [FixJ] = 2.9045 ng/µl #2 Ligation of the Pveg and FixJ T4 buffer: 2 µl T4 ligase: 1 µl Pveg: 25 ng/ 3.174ng/µl = 7.87= 8 µl FixJ: 25 ng/2.9045 ng/µl = 8.61= 9 µl 12.06.15 №1 PCR of the S.mutans 16s rRNA with synthesized primers S.mutans after gel extraction and genome isolation with Instagene Matrix 1 reaction 8 reactions DNA 15µl 120 µl Forward primer 5 µl 40 µl Reverse primer 5 µl 40 µl Master Mix 25 µl 200 µl Sterile Water 0 0 TOTAL 50 µl 400 µl Results: № 2 Single digest of the circular plasmid with EcoRI (NEB), SpeI (NEB), Aah1, and EcoRI (fermentas) 14.06.15 №1 S.mutans 16s rRNA PCR 1 reaction 8 reactions DNA 15µl 120 µl Forward primer 5 µl 40 µl Reverse primer 5 µl 40 µl Master Mix 25 µl 200 µl Sterile Water 0 0 TOTAL 50 µl 400 µl №2 Digest of the Pet 21 with NotI · DNA= 1000/240.8 ng/µl = 4.15 µl · NotI = 1µl · Cut smart = 5 µl · Sterile water = 39.85 µl №3 Digest of the GFP with NotI · DNA = 1000 ng/117.2 ng/µl = 8.53 µl · NotI = 1µl · Cut smart = 5 µl · Sterile water = 35.47 µl №4 Pveg and FixJ Ligation (2033 base pair) №5 Digest of the FixK2+rbs with SpeI (NEB) (PCR product digest) · DNA =1000ng/121.9 ng/µl = 8.2 µl · SpeI = 1µl · Cut smart = 5 µl · Sterile water = 35.8 µl №5 Digest of the FixK2+rbs with Aah1 (SibEnzyme) (PCR product digest) · DNA = 1000 ng/121.9 ng/µl = 8.2 µl · Aah1 = 1 µl · SEB buffer = 5µl · BSA= 0.5µl · Sterile water = 35.3 µl Results: The size of the FixK2+rbs (265 base pairs) do not coincide with the gel 15.06.15 №1 Solution on the day of the 15.06.2015 Transformation of the parts from Kit 2014 · Promoter FixK2 (Plate 1, 19G) – 250 base pair · RBS (Plate 4, 4G) – 15 base pairs · FixJ (Plate 1, 10N) – 1796 base pairs · RFP mCherry with double terminator (Plate 1, 13K) = 861 b.p · Promoter Veg (plate1, 20G) = №2 Genome extraction from S.mutans with Instagene Matrix №3 PCR of the 16S rRNA of the S.mutans Photo will be here 16.06.2015 № 1 Mini prep of of the parts pFixK2 = 78.03 ng/µl Rbs = 171.8 ng/µl FixJ = 124.5 ng/µl Rfp + double terminator = 82.75 ng/µl Promoter Veg = 42.20 ng/µl № 2 Double Digest of the FixK2 DNA : 12.82 µl Cut smart: 5 µl SpeI:0.5 µl EcoRI: 0.5 µl Sterile water: 38.18 µl Overall: 50 µl №3 Single digest of the FixK2 DNA: 12.82 µl SpeI: 1 µl Сut Smart Buffer: 5 µl Sterile Water: 31.18 µl № 4 Double Digest of the RBS DNA: 5.82 µl EcoRI: 0.5 µl XbaI : 0.5 µl Buffer: 5 µl Sterile water: 38. 18 µl № 5 Colony PCR 17.06.2015 №1 Gel extraction of the FixK2 and rbs FixK2 (double digest) = 4.128 ng/µl FixK2 (single digest) = 3.315 ng/µl Rbs = 3.051 ng/µl №2 Ligation of the FixK2 (double digest) + rbs FixK2 = 7µl Rbs = 9 µl T4 ligase = 1 µl T4 buffer = 2 µl Sterile water = 1 µl № 3 Ligation of the FixK2(single digest )+rbs in order to obtain linear plasmid FixK2 = 8 µl rbs = 9 µl T4 -ligase = 1 µl T4 buffer = 2 µl № 4. Double digest of 1st line -Pveg (EcorI-SpeI), 2nd line FixJ (EcorI-XbaI), 3rd line RFP (EcorI - XbaI): № 5. Transformation of (Fixk2 + rbs) was done, one tube. 18. 06.2015 Double check Transformation of [Fixk2 + rbs] We did not simultaneously perform digest and ligation since we wanted to check the work of the enzymes in double digest 2. Gel extraction of parts: Pveg, FixJ, RFP 3. Ligation reaction (rest parts): Pveg + FixJ Pveg +RFP. Reaction volumes for two reactions are the same: ul = µl Pveg = 24 ul FixJ/RFP = 48 ul Buffer = 8 ul ligase = 4 ul However, from now on we will use the the protocol DNA distribution according to 50 ng (insert) : 50 ng (backbone) notation 4. Chrm and Bacitracin plates were done (Bacitracin Mol. weight is 1422.71 g/mol and the concentration in stock (ex: 500 ml LB agar) should be 0.2 µM, while the conc. of bacitracin is 50 mg/ml): (50 mg/ml)/ 1422. 71 = 0.035 M in eppendorf tube 0.035 M x Y= 0.2 µM x 500 ml Y = 0.00286 ml = 2. 86 µl Chrm conc: 500 µl for 500 ml of LB agar 5. MinElute Reaction cleanup kit was obtained (to purify after digestion and go directly to transformation) Ethanol (220 ml) was added 6. Kit has arrived!!!!!) 7. Miniprep of LB broth (Fixk2+rbs) FixK2+ rbs = 217.7 ng/µl 19. 06. 2015 № 1 Transformation of the ligated parts 1- agarplate ; dCas9 under xylose (2015 Kit, plate=5, 8L) chloromphenicol resistant 2- agar plate: Anderson promoter (plate 1, 20 K) chloromphenicol resistant 3-agar plate: Anderson promoter (plate1, 22A) 4-agar plate: Anderson promoter (plate 1, 22 K) 5-agar plate; GFP (plate 4, 21J) 6-agar plate; Mukhtars part (plate 5, 24D) 7-agar plate; P veg+ FixJ 8-agar plate; Pveg+ RFP 9-agar plate; Pveg+ FixJ (20 µl) 10 - agar plate: Pveg + RFP (20 µl ) 2. PCR confirmation of the Fixk2+rbs ligation part obtained from miniprep 1- ladder, 2- FixK2, 3- FixK2+rbs, 4 - FixK2+ rbs Photo will be here Results; The size of the FixK2, FixK2 + rbs do not coincide with the right one. 3. Genome extraction from S.mutans by Instagene Genome Extraction and PCR PCR S.Mutans = 15 ul P1 = 5 ul P2 = 5 ul MM (Buffer) = 25 ul Fikx2 = 0.4 ul (on 3 tubes per 20 ul) VF2 = 6 ul VR = 6 ul MM = 30 ul water = 17.6 ul RESULTS: S.Mutans = No band amplification FixK2 = 600 bp part was shown as amplified Photo will be here! 20.06.2015 25.06.2015 № 1 PCR of the S.mutans. Number 2 plate (grown from single colony). Genome isolated by Instagene Matrix DNA = 15 ul Primer Forward = 5 ul Primer Reverse = 5 ul Master Mix = 25 ul № 2 PCR of the S.mutans ( colony taken from Namber 2 plate) DNA = 15 ul Primer Forward = 5 ul Primer Reverse = 5 ul Master Mix = 25 ul Results: The amplicon of the size 479 base pair is detected. First raw is the ladder, second is the amplicon which was PCR-ed with extension time 1 min, third is the amplicon with extension time with 14 seconds. №3 Negative control with the Bacillus Subtilis genome. Genome of the Bacillus was extracted with Instagene matrix 26.06.2015 30.06.2015 №1 Transformation of the FixK2 ( Kit 2015, Plate 1, 19G) №2 Restriction digest of the FixJ with the EcoRI and XbaI DNA = 1000ng/124.5ng/ul = 8 ul EcoRI = 1 ul XbaI = 1 ul Cut Smart = 5 ul Sterile Water = 35 ul Second raw after ladder is FixJ (already cut for gel extraction ) №3 Restriction Digest of the Pveg with EcoRI and SpeI DNA = 1000 ng/42.20ng/ul = 24 ul EcoRI = 1 ul SpeI = 1 ul Cut Smart = 5 ul Sterile Water = 19 ul №4 Gel extraction of the FixJ and Pveg FixJ = 2.217 ng/ul Pveg =2.892 ng/ul №5 Ligation was performed in two ways. DNA was taken from gel extraction Pveg= 9 ul FixJ = 8 ul T4 -ligase = 1 ul T4 -buffer = 2 ul 2. DNA was taken directly from digestion solution without clean up Pveg = 8.5 ul FixJ = 8.5 ul T4 ligase= 1 ul T4 buffer= 2 ul 07/07/15 Activity № 1 Results: Digest = sequential (1 line): 250 bp = Fixk2 PCR gradient: 4-11 line, 580 bp cmv promoter, actual size = 900 bp, digest of cmv was successful but , pcr shows that primers are impaired, since Master Mix and water were new and clean, dna was the same as for digest. 8.07.2015 Activity № 1. Sequential Digest of the Pveg with SpeI (sib enzyme) and EcoRI (fermentas) Pveg = 12.5 ul SpeI (sib) = 1 ul BSA = 0.5 ul Seb buffer = 5 ul Sterile water = 31 ul Incubation for 2 hours at 37 degree C + NaCl = 1 ul (5 M) +BSA (0.5 ul) +1 ul EcoRI (Fermentas) Results: Pveg of the size 237 base pair was seen on the agarose gel 9.07.2015 Activity №1. Transformation FixK2+rbs Cmv+neomycin GFP Anderson promoter Anderson promoter Anderson promoter Activity № 2. Digest of the RFP mcherry+ double terminator with Invitrogen EcoRI and XbaI Results: The second line shows that there are two bands that are located very close to each other. This result suggests that upper band is the uncut plasmid, while the lower band is the one that was successfully digested. However, there is still doubt is it cut with one enzyme or both? Activity № 3. Gel extract of the Pveg that was sequentially digested with SpeI (sib enzyme) and EcoRI (fermentas) Pveg = 30.78 ng/ul mcherryRFP + double terminator (digested with EX from Invitrogen) = 9.36 ng/ul Activity № 4. Ligation of the Pveg+ mcherryRFP with double terminator 10x T4 DNA Ligase Buffer = 2 ul T4-ligase = 1 ul Pveg = 150 ng/30ng/ul = 4.87 ul RFP = 50 ng/9.36 ng/ul = 5.34 ul Sterile water = 6.79 ul 10.07.2015 Activity № 1. Checking of the Ligation of the Pveg+FixJ mini prep product by making sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas) Results: There are two bands. One is the 2500 bp. Another is 2000 base pair. However, if Pveg+ FixJ ligation have worked there would be also uncut plasmid after digestion of size 4500. So, it was decided to make single digest with EcorI in order to check if there would be a liniarized plasmid of 4500 base pair size. Activity №2. Single digest of the Pveg+FixJ with the EcoRI (Neb enzyme) DNA= 1000 ng/94.69 ng/ul = 10.56 ul Cut smart = 5 ul EcoRI = 1 ul Sterile water = 33.44 ul Overall: 50 ul Results: First raw is ladder, second is plasmid after ligation of Pveg+FixJ, third is Pveg+FixJ plasmid cut with EcoRI. The results shows that there is no linearized plasmid with the 4500 base pair after ligation. So ligation did not work. Activity №3. Transformation Double Terminator =2014 Kit, plate 1, 3D, chloromphenicol RBS = 2014 Kit, plate 4, 4G, chloromphanicol Pveg= 2014 Kit, plate 1, 20G, chloromphenicol Ligation product= Pveg + mcherry RFP (cut was done by EX of Invitrogen)