Team:UPO-Sevilla/Notebook
JANUARY
| Monday | Tuesday | Wednesday | Thursday | Friday |
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| 1 | ||||
| 4 | 5 | 6 | 7 | 8 |
| 11 | 12 | 13 | 14 | 15 |
| 18 | 19 | 20 | 21 | 22 |
| 25 | 26 | 27 | 28 | 29 |
APRIL
| Monday | Tuesday | Wednesday | Thursday | Friday |
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| 1 | ||||
| 4 | 5 | 6 | 7 | 8 |
| 11 | 12 | 13 | 14 | 15 |
| 18 | 19 | 20 | 21 | 22 |
| 25 | 26 | 27 | 28 | 29 |
JULY
| Monday | Tuesday | Wednesday | Thursday | Friday |
|---|---|---|---|---|
| 1 | ||||
| 4 | 5 | 6 | 7 | 8 |
| 11 | 12 | 13 | 14 | 15 |
| 18 | 19 | 20 | 21 | 22 |
| 25 | 26 | 27 | 28 | 29 |
JANUARY
7th January
Biofilm Module
Prepare plates with LB+Km and plate the strain with pSB1K3 (E. coli DH5α)
Back to the calendar8th January
Biofilm Module
Plate the strains of E. coli DH5α with pMPO364 (nasF and nahR-Psal), JB3Tc19::PleD (pleD*), pMPO52 (Pm) and pMRB11(yhjH), and P. putida KT2442 (lapG)
Back to the calendar11th January
Biofilm Module
Inocula of pSB1K3 in 15 mL of LB+Km (kanamycin)
Inocula of each strain in 3 mL of LB+specific antibiotic and incubate overnight
Back to the calendar12th January
Biofilm Module
Minipreps of pSB1K3
Minipreps and measure the concentration of the cultures
Back to the calendar13th January
Biofilm Module
14 first primers have arrived. Resuspend them, dilute until 10 μM and storage at -20ºC
PCR of pMPO364 and pMRB11 [45ºC]
PCR of pJBeTc19::pleD and Pseudomonas putida KT2442 chromosome [60ºC]
Electrophoresis of both PCR
Back to the calendar14th January
Biofilm Module
Repeat the PCR at 45ºC and at 60ºC
Electrophoresis of both PCR
Purify the 5 PCR products (lapG, nahR-Psal, nasF, pleD*, yhjH) and store them at -20ºC
Back to the calendar15th January
Biofilm Module
Digest the purified PCR products and pSB1K3 where we will clone the PCR products
Electrophoresis of the digested PCR products and vector and cut a slice of the gel that contain the fragment of interest and purify
Plate the strain with pMPO1035 (xylS2)
Back to the calendar18th January
Biofilm Module
Ligation the vector with each PCR product
Put one colony of pMPO1035 in 3 mL of LB+Cm (Chloramphenicol)
Back to the calendar19th January
Biofilm Module
Transformation of competent cells, E. coli DH5α, with the ligations
Miniprep and measure the concentration of pMPO1035
Back to the calendar20th January
Biofilm Module
Inocula of each transformation event in 3 mL of LB+Km
PCR of pMPO1035
Back to the calendar21th January
Biofilm Module
Miniprep the plasmid DNA of the transformations
Diagnostic digest the plasmids with PstI and XbaI
Electrophoresis of the digestions and the PCR. All the fragments had the correct length
Purify the PCR of xylS2 and digest with PstI and XbaI
Purify the digestion of xylS2 and ligation with pSB1K3
Back to the calendar22th January
Biofilm Module
Transformation of competent cells, E. coli DH5α, with the ligation
Back to the calendar25th January
Biofilm Module
Repeat the ligation of xylS2 with the vector, because the plates were contaminated
Back to the calendar26th January
Biofilm Module
Send to sequence 4 constructions (lapG, nahR-Psal, nasF, yhjH)
Transformation of competent cells, E. coli DH5α, with the ligation
Back to the calendar27th January
Biofilm Module
The transformation was unsuccessful, so we did a electrophoresis of the vector and the insert (xylS2) to test that we have DNA in the sample
Repeat the ligation of xylS2 with the vector
Dilute until 10 μM the primers that we use to do a overlap extension PCR
Back to the calendar28th January
Biofilm Module
Test the received sequences of lapG, nahR-Psal, nasF and yhjH. All of them were correct
Transformation of competent cells, E. coli DH5α, with the ligation and the tested constructions. Now we name these constructions as pMRB120 (nahR-Psal), pMRB121 (lapG), pMRB122 (yhjH) and pMRB123 (nasF)
Digest pMRB121, pMRB122 and pMRB123 as vector and pMRB120 as insert
Back to the calendar29th January
Biofilm Module
Electrophoresis of the digestions and cut a slice that contains the fragment of interest and purify
Back to the calendarFEBRUARY
1st February
Biofilm Module
Inocula of each transformation and xylS2 in 3 mL of LB+Km
Ligation of yhjH, lapG and nasF with nahR-Psal
Back to the calendar2nd February
Biofilm Module
Miniprep and diagnostic digestion of xylS2
Storage the pMRB120, pMRB121, pMRB122 and pMRB123 strains at -80ºC
Transformation of competent cells, E. coli DH5α, with the ligations
Send to sequence the constructions of xylS2 and pleD*
Back to the calendar3th February
Biofilm Module
Take biomass of the transformations and plate onto other plate, because there were too many cells on the plates
First part of overlap extension PCR of Pm
Back to the calendar4th February
Biofilm Module
Inocula of the transformation in 3 mL of LB+Km
Electrophoresis of PCR of Pm, but it did not go well
Repeat the PCR of Pm
Back to the calendar5th February
Biofilm Module
Miniprep of nahR-Psal-lapG, nahR-Psal-yhjH and nahR-Psal-nasF
Diagnostic digest of these plasmids
Test the received sequences of xylS2 and pleD*. Only xylS2 is correct, so do a transformation to introduce the plasmid, now we name this construction as pMRB124
Back to the calendar8th February
Biofilm Module
Inocula of xylS2 strain in LB+Km
Transformation with nahR-Psal-yhjH V, nahR-Psal-lapG IV and nahR-Psal-nasF II. Now, we name the constructions as pMRB125, pMRB126 and pMRB127, respectively
Electrophoresis of PCR product, but only Pm RM 1 (the fragment of Pm amplified with the primer reverse and mutagenic forward 1) did not go well
Back to the calendar9th February
Biofilm Module
Inocula of each transformation in LB+Km
Storage the pMRB124 strain at -80ºC
Repeat the PCR of Pm RM 1
Electrophoresis of the PCR
Back to the calendar10th February
Biofilm Module
Storage the pMRB125, pRMB126 and pMRB127 strains at -80ºC
Purify all the PCR products of Pm
Digest pMRB127 with EcoRI and SpeI as insert
Ligation of the vector with pleD*, and lapG and yhjH with nahR-Psal-nasF
Back to the calendar11th February
Biofilm Module
Electrophoresis of the digestion of pMRB127, cut the slice that contains the fragment of interest and purify
Transformation with the ligations
Overlap extension PCR of Pm1, Pm2 and Pm3, and PCR of Pm RM1 and Pm (In)
Electrophoresis of the overlap extension PCR
Back to the calendar12th February
Biofilm Module
Electrophoresis of Pm RM1 and Pm FM1 to compare the concentrations. Dilute 10 times and repeat the overlap extension PCR
Electrophoresis of the overlap extension PCR of Pm1. It does not go well
Back to the calendar15th February
Biofilm Module
Inocula of nahR-Psal-nasF-lapG and nahR-Psal-nasF-yhjH (complex devices), pUC18Sfi;miniTn7BBGm and pleD* in LB
Repeat overlap extension PCR of Pm1
Back to the calendar16th February
Biofilm Module
Miniprepion of the plasmid DNA of the cultures
Diagnostic digestion of pleD* and complex devices
Electrophoresis of the diagnostic digestions and the PCR of Pm1
Back to the calendar17th February
Biofilm Module
Transformation with pleD* and complex devices. Now, we name the constructions as pMRB145, pMRB129 and pMRB130, respectively
Digest Tn7 and the vector as vector and Pm, Pm1, Pm2, Pm3, nahR-Psal-nasF-lapG and nahR-Psal-nasF-yhjH
Ligation of the vector with Pm, Pm1, Pm2 and Pm3 (Pm vectors), and Tn7 with nahR-Psal-nasF-lapG and nahR-Psal-nasF-yhjH (Tn7 devices)
Back to the calendar18th February
Biofilm Module
Inocula of each transformation in LB+Km
Transformation with the ligations
Back to the calendar19th February
Biofilm Module
Miniprep of pleD* and complex devices
Storage pMRB145, pMRB129 and pMRB130 at -80ºC
Back to the calendar22th February
Biofilm Module
Incocula of each transformation in LB
Plate the strains with pMRB124 (xylS2) and pTNS2 (helper of Tn7)
Digest pMRB129 and pMRB130 as insert
Back to the calendar23th February
Biofilm Module
Miniprep of the cultures
Electrophoresis of the digestions, cut a slice that contains the fragment of interest and purify
Inocula of 124 and pTNS2 in 3 mL of LB
Back to the calendar24th February
Biofilm Module
Diagnostic digestion of Pm vectors and Tn7 devices
Electrophoresis of the diagnostic digestions of Pm vectors and Tn7 devices
Miniprep of the cultures of 124 and pTNS2
Send to sequence the constructions of Pm
Back to the calendar25th February
Biofilm Module
Repeat the transformation with Tn7 devices. Now, we name the constructions as pMRB133 (Tn7-nahR-Psal-nasF-lapG) and pMRB134 (Tn7-nahR-Psal-nasF-yhjH)
Digest 124, 120 and pSB1K3 as vector
Ligation of 124 (v) with 120 (i) and 127 (i)
Inocula of pMRB125 and pMRB126 in 3 mL of LB
Back to the calendar26th February
Biofilm Module
Segregate the plates of 133 and 134
Miniprep of the cultures of 125 and 126
Heat-shock transformation with the ligations
Digest 125 and 126 as insert
Glycerol Module
PCR glpF (primers 1-2, 3-4 and 1-4). Tª annealing 53ºC
Electrophoresis PCR glpF. Fragment amplified with primers 3-4 is OK
Back to the calendar29th February
Biofilm Module
Inocula of 133 and 134 in 5 mL of LB and 124+120 in 3 mL of LB
Segregate the plate of 124+127
Heat-shock transformation with the constructions of Pm. Now, we name the constructions as pMRB137 (Pm), pMRB138 (Pm1), pMRB139 (Pm2) and pMRB140 (Pm3)
Electrophoresis of the digestions of 125 and 126, cut a slice of gel that contains the fragment of interest and purify
Back to the calendarMARCH
1st March
Biofilm Module
Miniprep of the cultures
Storage pMRB133 and pMRB134 at -80ºC
Inocula of 124+127 in 3 mL of LB, and each construction of Pm in 5 mL of LB
Glycerol Module
Concentration measurement of glpF amplified with primers 1-4, using Nanodrop
PCR glpF Repetition (primers 1-2, using genomic DNA and fragment amplificated with primers 1-4 as templates, and primers 3-4). Tª annealing 56ºC.
Back to the calendar2nd March
Biofilm Module
Miniprep of the culture
Segregate the plates of the transformations
Digest and electrophoresis of 124+127
Ligation Tn7 with 125 (i) and 126 (i)
Glycerol Module
Electrophoresis PCR glpF
Back to the calendar3th March
Biofilm Module
Storage pMRB137, pMRB138, pMRB139 and pMRB140 at -80ºC
Transformation of the ligations and pleD*. Now, we name the construction of pleD* as pMRB145
Digest 124 as vector
Electrophoresis of the digestion, cut a slice of gel that contains the fragment of interest and purify
Ligation 124 (v) with 120 (i) and 127 (i), and Tn7 with 125 (i) and 126 (i)
Glycerol Module
PCR glpF with primers 1-2, using genomic DNA as template. Tª annealing 58ºC
Back to the calendar4th March
Biofilm Module
Segregate the plate of the transformation
Heat-shock transformation with the ligations
Plate the strain with pMRB1 (vector that contains the protein fusion gen lapZ-gfpmut3)
Glycerol Module
Electrophoresis PCR glpF with primers 1-2
Back to the calendar7th March
Biofilm Module
Inocula of 145 in 5 mL of LB
Inocula of pMRB1,124+120 and 124+127 in 3 mL of LB
Glycerol Module
PCR glpF with primers 1-2. Tª annealing 59ºC
Electrophoresis PCR glpF
Back to the calendar8th March
Biofilm Module
Storage pMRB145 at -80ºC
Miniprep of the cultures
Digest 145 as vector and 137, 138, 139 and 140 as insert
Electrophoresis of the digestions, cut a slice of gel that contains the fragment of interest and purify
Diagnostic digestion of the pMRB124 constructions
Glycerol Module
PCR glpF with primers 1-2. Tª annealing 61ºC
Back to the calendar9th March
Biofilm Module
Electrophoresis of the diagnostic digestions
Digest pMRB1 as vector
Repeat the ligation of Tn7 with 125 (i) and 126 (i), and 145 (v) with 120 (i) and 127 (i)
Segregate the plate of 124+127. Now, we name this construction as pMRB146
Glycerol Module
Electrophoresis glpF
Back to the calendar10th March
Biofilm Module
Transformation with the ligations
Electrophoresis of the digestion of pMRB1 (v), cut a slice of gel that contains the fragment of interest and purify
Ligation of pMRB1 (v) with the four Pm (i)
Glycerol Module
PCR glpF. Tª annealing 64ºC
Back to the calendar11th March
Biofilm Module
Transformation with the ligations
Plate the strain with 128
Digest of pMRB125 and pMRB126 as insert
Glycerol Module
Electrophoresis PCR glpF
PCR glpF 1-2:
1. 1 μl genomic DNA + 1 μl DMSO. 67ºC
2. 1 μl genomic DNA diluted 1/10. 67ºC
Back to the calendar14th March
Biofilm Module
Inocula of 145+120, 145+127, Pm contructions, 128 and 146
Diagnostic digestion of 145+120 and 145+127
Ligation of Tn7 with 120 (i) and 127 (i), and 124 (v) with 120 (i)
Glycerol Module
Electrophoresis PCRs glpF
Mix of PCRs glpF (45 μl DMSO 67ºC + 45 μl genomic diluted 1/10 67ºC + 45 μl 64ºC
Back to the calendar15th March
Biofilm Module
Miniprep of the cultures and 128 digest as vector
Diagnostic digestion of the Pm constructions
Storage pMRB146 at -80ºC
Transformation with the ligations
Glycerol Module
Geneclean of the band of the fragment of glpF amplified with primers 1-2, using the mix created the day 1/7
Back to the calendar16th March
Biofilm Module
Electrophoresis of the diagnostic digestions
Electrophoresis of the digestions of 125 (i) and 126 (i), cut a slice of gel that contains the fragment of interest and purify
Ligation of Tn7 with 120, 125 and 126, and 145 (v) with 120 (i)
Inocula of Tn7+127 and 124+120
Glycerol Module
Electrophoresis of the geneclean of glpF 1-2 and concentration measurement using Nanodrop
Back to the calendar17th March
Biofilm Module
Miniprep of the cultures
Diagnostic digestion of 124+120 and Tn7+127
Transformation with the ligations
Glycerol Module
Overlap extension PCR glpF (5μl fragment 1-2 + 2,22 μl fragment 3-4 diluted 1/100). 67ºC
Electrophoresis overlap PCR glpF
Back to the calendar18th March
Biofilm Module
The transformations with Tn7 devices didn't work, so plate Tn7 strain again
Transformation with 145+127. Now, we name this construction as pMRB147
Glycerol Module
Geneclean overlap PCR glpF
Electrophoresis geneclean overlap PCR glpF
Back to the calendar21th March
Biofilm Module
Inocula of Tn7, 147, 145+120 and 145+127
Repeat the ligation of pMRB1 (v) with 137 (i)
Back to the calendar22th March
Biofilm Module
Miniprep of the cultures
Diagnostic digestion of 145+127 and 145+120
Electrophoresis of pMRB128 (v), cut a slice of gel that contains the fragment of interest and purify
Transformation with pMRB1+137 and Tn7+127
Ligation of Tn7 (v) with 125 (i)
Back to the calendar23th March
Biofilm Module
Inocula of pMRB1+137
Transformation with Tn7+pMRB125
Electrophoresis of the diagnostic digestion of 145+120
Back to the calendar24th March
Biofilm Module
Minipreps and diagnostic digestion of 1+137
Segregate the plate of Tn7+127
Digestion of 145 and 147 as an insert
Ligation of Tn7 (v) with 125 (i) and 126 (i), and 145 (v) with 120 (i)
Back to the calendar25th March
Biofilm Module
Transformation of the ligations
Electrophoresis of the digestions, cut a slice of gel that contains the fragment of interest and purify
Diagnostic digestion of pMRB1+137/138/139/140
Back to the calendar28th March
Biofilm Module
Inocula of Tn7+127. Now, we name this construction as pMRB151
The transformations did not go well
Back to the calendar29th March
Biofilm Module
Ligation of 120 (v) with 124 (i), Tn7 (v) with 125 (i), 147 (i) and 126 (i), pMRB1 (v) with 137-140 (i) and 145 (v) with 120 (i)
Store at -80ºC and minipreps of pMRB151
Back to the calendar31th March
Biofilm Module
Inocula of 1+137/138/139/140, 120+145,120+124 and Tn7+147
Segregate the transformation of Tn7+126
Back to the calendarAPRIL
1st April
Biofilm Module
Minipreps and diagnostic digestion of the 1+137/138/139/140, 120+124, 145+120 and Tn7+147
Electrophoresis of the diagnostic digestion
Back to the calendar4nd April
Biofilm Module
2nd diagnostic digestions of 1+137/138/139/140
Inocula of 124,135 and 136
Transformation of 145+120 and Tn7+147. Now, we name these constructions as pMRB135 and pMRB136, respectively
Glycerol Module
Overlap PCR glpF 71ºC and electrophoresis
Back to the calendar5th April
Biofilm Module
Storage pMRB135 and pMRB136 at -80ºC
Inocula of KT2442
Digest 135,145 and 130 as insert and Tn7 as vector
Measure the concentration of pTNS2, pMRB136 and pMRB134>
Glycerol Module
Precipitation of overlap PCR of glpF and concentration measurement using Nanodrop
Back to the calendar6th April
Biofilm Module
Electrophoresis of digestion of 135, 145, 130 and Tn7, cut a slice of gel that contains the fragment of interest and purify
Plate the strains with pMRB136 and pTNS2
Electroporation of KT2442 with 136 and 134
Glycerol Module
Overlap PCR of glpF 71ºC (again) and electrophoresis. There is nothing!
Back to the calendar7th April
Biofilm Module
Inocula of pTNS2 and 136
Ligation of Tn7 with 126 and 135
Colony PCR of the electroporations
Glycerol Module
PCR glpF 1-2:
1. 1 μl genomic DNA + 1 μl DMSO. 67ºC
2. 1 μl genomic DNA diluted 1/10. 67ºC
Electrophoresis PCRs glpF 1-2
Back to the calendar8th April
Biofilm Module
Minipreps of pTNS2 and 136
Transformation of the ligations
Electrophoresis of the colony PCR
Glycerol Module
Geneclean of the PCRs 1-2 and nanodrop measurement
Back to the calendar11th April
Biofilm Module
Inocula of Tn7+126 and Tn7+135
Ligation of 1 (v) with 138 (i), 124 (v) with 120 (i) and Tn7 (v) with 120 (i) and 125 (i)
Glycerol Module
Electrophoresis geneclean PCR glpF 1-2
Overlap PCR glpF (using fragment 1-2 obtained the day 24/1)
Electrophoresis of overlap PCR glpF
Back to the calendar12th April
Biofilm Module
Minipreps and diagnostic digestion of the cultures
Transformation of the ligations
Glycerol Module
glpF fragment (overlap PCR precipitated the day 31/1) digestion using XbaI and PstI restriction enzymes
Back to the calendar13th April
Biofilm Module
Inocula of 1+138, 124+120, and Tn7+125. Now ,we name 124+120 as pMRB152
Ligation of Tn7 (v) with 130 (i), 145 (i), 146 (i) and 120 (i)
Glycerol Module
Heat inactivation restriction enzymes (20’ 80ºC)
Measurement of the concentration of glpF digested using nanodrop
Back to the calendar14th April
Biofilm Module
Minipreps of the cultures
Storage pMRB152 at -80ºC
Diagnostic digestion and electrophoresis of 1+137/138/139/140, Tn7+125, Tn7+135 and Tn7+126
Transformation with Tn7+120, Tn7+145, Tn7+130, Tn7+146 and Tn7+125
Glycerol Module
Digested pSB1K3 obtaining and electrophoresis
Back to the calendar15th April
Biofilm Module
Measure the concentration of 136 and 134
Diagnostic digestions of 1+138, Tn7+126 and Tn7+135
Plate the strain with pTNS2
Glycerol Module
Geneclean of the fragment of the plasmid that will be used to clone glpF and measurement of the concentration using nanodrop
Back to the calendar18th April
Biofilm Module
Inocula of KT2442, pTNS2, Tn7+145, Tn7+130, Tn7+146, Tn7+120 and Tn7+125. Now, we name Tn7+125 as pMRB128, Tn7+130 as pMRB164 and Tn7+145 as pMRB165
Electrophoresis of the diagnostic digestions
Glycerol Module
Ligation of glpF to pSB1K3
Back to the calendar19th April
Biofilm Module
Electroporation of KT2442 with 164, 165, 134 and 136
Minipreps of pTNS2, Tn7+146 and Tn7+120
Storage pMRB128, pMRB164 and pMRB165 at -80ºC
Transformation of 1+Pms. Now, we name 1+Pm, 1+Pm1, 1+Pm2 and 1+Pm3 as pMRB166, pMRB167, pMRB168 and pMRB169
Glycerol Module
Transformation of the ligation to bacteria (E. coli DH5α)
Back to the calendar20th April
Biofilm Module
Colony PCR of the electroporations and electrophoresis
Diagnostic digestion of Tn7+146 and Tn7+120
Segregate the positive candidates of electroporations
Plate KT2442-Tn7
Inocula of 166, 167, 168 and 169
Glycerol Module
White colonies obtained. 3 inocula of the colonies of pSB1K3-glpF
Back to the calendar21th April
Biofilm Module
Inocula of KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165
Electrophoresis of Tn7+146 and Tn7+120
Diagnostic digestion of 120 and 146
Storage at -80ºC and minipreps of pMRB166-169
Glycerol Module
Minipreps of the plasmids
Digestion of the plasmids
Back to the calendar22th April
Biofilm Module
Digest 152 as insert
Electroporation of KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165 with pCdrA
Glycerol Module
Electrophoresis of the digested samples. No good results. There was no a band with the size of glpF
Back to the calendar25th April
Biofilm Module
Plate the strains KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165
Inocula for fluorimetry
Electrophoresis of 152 (i), cut a slice of gel that contains the fragment of interest and purify
Glycerol Module
PCR of the rest of colonies of the plaque of the ligation of glpF to pSB1K3, using Taq polymerase
Back to the calendar26th April
Biofilm Module
Digest pSB1K3 as vector
Inocula of KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165
Fluorimeter
Glycerol Module
Electrophoresis of the PCR of glpF. There are positive clones (numbers 6, 8, 11, 12 and 14)
Back to the calendar27th April
Biofilm Module
INSTANT curves of growth and biofilm of KT2442-Tn7/T134/T136/T164/T165
Failed fluorimeter
Glycerol Module
Inocula of the positive clones of pSB1K3-glpF. 2 inocula of 8 and 12 (they will be sequenced)
Back to the calendar28th April
Biofilm Module
Dye and measure the plates of INSTANT curves
Digest 120 as insert
Ligation Tn7+146 and Tn7+152
Glycerol Module
3 flasks dropped off in the incubator (6, one of the 8 and 11)
Freeze bacteria (DH5α with pSB1K3-glpF8, pSB1K3-glpF12 and pSB1K3-glpF14)
Minipreps of pSB1K3-glpF8 and pSB1K3-glpF12
Back to the calendar29th April
Biofilm Module
Transformation of the ligations
Plate 128, 133, KT2442-Tn7/T136/T165 and KT2442, KT2442 lapG- and KT2442 ΔbifA
Back to the calendarMAY
2st May
Biofilm Module
Inocula of Tn7+146, Tn7+152, KT2442 lapG-, 128 and 133
Plate all the KT2442 with pCdrA, KT2442 ΔfleQ and all the variants of KT2442 of pleD*
Electrophoresis of 120 (i), cut a slice of gel that contains the fragment of interest and purify
Back to the calendar3nd May
Biofilm Module
Minipreps and measure the concentration of 128 and 133
Minipreps and diagnostic digestion of Tn7+146 and Tn7+152
Electroporation of KT2442 lapG- with 128 and 133
Glycerol Module
Concentration measurement of plasmids 8 and 12 using nanodrop. Send to secugen to sequence
Back to the calendar4th May
Biofilm Module
Inocula for curves and fluorimeter
Electrophoresis of the diagnostic digestions
Dilutions of fluorimeter and curves of KTs
Glycerol Module
Digestion of the plasmids 8 and 12 with XbaI and PstI restriction enzymes
Back to the calendar5th May
Biofilm Module
Dye and measure the curves of pleD*
Take the data of the fluorimeter
Glycerol Module
Electrophoresis of the digestions
Spread bacteria (frozen at -80ºC) with plasmids 8 and 12 on agar plates, and let them grow at 37ºC
Back to the calendar6th May
Biofilm Module
Plate the variants of KT2442 with yhjH
Transformation of Tn7+120
Glycerol Module
Spread the bacteria grown at 37ºC on other plaques and let them grow at room temperature
Back to the calendar9th May
Biofilm Module
Inocula of KT2442-Tn7/T134/T164 and Tn7+120
Transformation with Tn7+146 and Tn7+152. Now, we name these strains as pMRB159 and pMRB170, respectively
Glycerol Module
Inocula using the plaques of the day 3/3. One of each plasmid (8 and 12)
Back to the calendar10th May
Biofilm Module
Minipreps and diagnostic digestion of Tn7+120
Dilutions of yhjH and put the curves for 20 h
Segregate KT2442 lapG--T128/T133
Glycerol Module
Wrong results of the sequencing due to a confusion with the primers used
Minipreps of pSB1K3-glpF8 and pSB1K3-glpF12
Back to the calendar11th May
Biofilm Module
Dye and measure the plates of yhjH
Inocula of 159 and 170
Electrophoresis of the diagnostic digestion
Colony PCR of KT2442 lapG--T128/T133
Transformation with Tn7+120
Glycerol Module
Digestion of plasmids pSB1K3-glpF8, pSB1K3-glpF12 and pSB1K3 with XbaI and PstI restriction enzymes
Back to the calendar12th May
Biofilm Module
Minipreps of the cultures
Storage pMRB159 and pMRB170 at -80ºC
Inocula of Tn7+120
Glycerol Module
Electrophoresis of the digested samples. pSB1K3-glpF8 is OK!
Back to the calendar
13th May
Biofilm Module
PCR of pMRB1 to get the gene gfp
Electrophoresis of the colony PCRs
Segregate Tn7+120
Back to the calendar16th May
Biofilm Module
Inocula of pleD* constructions in Tn7 and KT2442 lapG--T128
Inocula of Tn7+120
Colony PCR and electrophoresis of KT2442 lapG--T133
Back to the calendar17th May
Biofilm Module
Storage KT2442 lapG--T128 at -80ºC
Dilutions of pleD* curves
Plate KT2442 lapG- and KT2442 ΔbifA
Inocula of KT2442
Inocula of KT2442 and KT2442-Tn7/T134/T136/T164/T165 for Congo Red and curves
Glycerol Module
Send pSB1K3-glpF8 to Secugen to be sequenced
Back to the calendar18th May
Biofilm Module
Dye and measure the pleD* plates
Plate the strains with 134, 136, 164, 170, 159 and Tn7
Inocula of KT2442-Tn7/T165 for curves
Inocula of KT2442 lapG- and KT2442 ΔbifA
Plates of Congo Red
Curves of 134, 164 and KT2442
Glycerol Module
pSB1K3-glpF8 digestion using XbaI and PstI restriction enzymes
Back to the calendar19th May
Biofilm Module
Electrophoresis of the PCR of gfp
Curves of 165
Electroporation of KT2442 lapG- and ΔbifA with Tn7, 128 and 133
Dye and measure the plates of 134, 164 and KT2442
Let more time the plates with Congo Red
Glycerol Module
Electrophoresis of the digested sample and purification of the band of glpF
Back to the calendar20th May
Biofilm Module
Dye and measure the plates of 165 at 17 h and 20 h
Take a photo of the plates with Congo Red
Let the plates with Congo Red at RT overnight
Glycerol Module
Electrophoresis of the geneclean. OK!
Back to the calendar23th May
Biofilm Module
Inocula of 170, 159 and Tn7
Electroporation of KT2442 with 159, 170, 128 and 133
Minipreps and measure the concentration of 170, 159 and Tn7
Digestion of 164 and 148
PCR of gfp
Electrophoresis of the digestions and PCR
Glycerol Module
Repeat pSB1K3-glpF8 digestion and geneclean
Take out frozen bacteria with pSB1K3-glpF8 and let them grow in a liquid medium at 37ºC
Back to the calendar24th May
Biofilm Module
Colony PCR adn electrophoresis of the electroporations
Minipreps and measure the concentration of 170, 159 and Tn7
Plate all the strains with pCdrA and the same strains withour pCdrA (fluorimeter and Congo Red)
Inocula of KT2442-T170/T159/T128, lapG--Tn7 and ΔbifA-T128
Glycerol Module
Put the flask at 4ºC of the bacteria growing in a liquid medium
Back to the calendar25th May
Biofilm Module
Inocula of the plates of yesterday
Colony PCR and electrophoresis of KT2442 lapG--Tn7/T133, ΔbifA-T128/T133 and KT2442-T133
Storage KT2442-T170/T159/T128, lapG--Tn7 and ΔbifA-T128 at -80ºC
Glycerol Module
Results of the sequentiation of glpF. IT IS OK!!!
Centrifugation of the flask and freeze the pellet at -80ºC
Back to the calendar26th May
Biofilm Module
Assay of the fluorimeter with all the constructions and ΔfleQ
Plate of Congo Red with KT2442, Tn7/T134/T136/T164/T165
Colony PCR and electrophoresis of KT2442-T133, KT2442 lapG--T133 and KT2442 ΔbifA-T133
Glycerol Module
Electrophoresis of the purified band of pSB1K3-glpF8
Back to the calendar27th May
Biofilm Module
Get the results of the fluorimeter
Let the plates with Congo Red 24 h more
Electrophoresis of the digestion of 164, cut a slice of gel that contains the fragment of interest and purify
Glycerol Module
Miniprep of pSB1K3-glpF8 (frozen pellet at -80ºC)
Back to the calendar30th May
Biofilm Module
Plate KT2442, KT2442-T134/T164, KT2442-Tn7/T128 and lapG--Tn7/T128
A labmate got the plates with Congo Red and took a photo of >
Glycerol Module
pMRB151 (mini-Tn7-nahR/Psal/nasF) digestion with SpeI and PstI and electrophoresis
Back to the calendar31th May
Biofilm Module
Inocula of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Plate pMRB133
Glycerol Module
Geneclean of pMRB151 digested. Purify the fragment that will be used for the ligation
Back to the calendarJUNE
1st June
Biofilm Module
Dilutions and prepare curves of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Inocula of KT2442-Tn7/T128 and lapG--Tn7/T128
Glycerol Module
Electrophoresis of the geneclean and measurement of the concentration using nanodrop
Ligation of pMRB151 and glpF (1:3 proportion)
Back to the calendar2nd June
Biofilm Module
Dye and measure the plates of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Dilutions and prepare curves of KT2442-Tn7/T128 and lapG--Tn7/T128
Inocula of 133
Glycerol Module
Transformation of half the volume of the ligation
Back to the calendar3th June
Biofilm Module
Dye and measure the plates of KT2442-Tn7/T128 and lapG--Tn7/T128
Miniprep and measure the concentration of 133
Glycerol Module
There is nothing!
Back to the calendar6th June
Biofilm Module
Plate KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Diagnostic digestion and electrophoresis of 133
Glycerol Module
Transformation of the rest of the ligation sample
Back to the calendar7th June
Biofilm Module
Inocula of KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Electroporation of KT2442, KT2442 lapG- and ΔbifA with pMRB133
Glycerol Module
There is nothing!
Digestion of the geneclean of pMRB151
Back to the calendar8th June
Biofilm Module
Dilutions and prepare curves of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Colony PCR and electrophoresis of 133 electroporations
Inocula of KT2442/lapG-/ΔbifA-T133
Glycerol Module
Electrophoresis of the digestion
Back to the calendar9th June
Biofilm Module
Dye and measure the plates of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Glycerol Module
Geneclean of digested pMRB151. Purify the fragment that will be used for the ligation
Back to the calendar10th June
Biofilm Module
Storage KT2442/lapG-/ΔbifA-T133 at -80ºC
Glycerol Module
Electrophoresis of the purified band. There is nothing!
Back to the calendar13th June
Glycerol Module
Spread bacteria with pMRB151on agar plates and let them grow at 37ºC
Back to the calendar15th June
Glycerol Module
Minipreps of pMRB151 and electrophoresis
Digestion of pMRB151 A, B and C (diagnostic digestions)
Digestion of pMRB151 C (for cloning)
Digestion of the miniprep of pSB1K3-glpF8
Electrophoresis of all digestions
Repeat diagnostic digestions
Back to the calendar17th June
Glycerol Module
Geneclean pSB1K3-glpF8 digested
pSB1K3-glpF8 digestion with EcoRI and XbaI
Spread bacteria with pSB1K3-glpF8 and incubate at room temperature
Back to the calendar20th June
Glycerol Module
Electrophoresis
5 inocula (pSB1K3-glpF8 A, B , C, D and E)
Back to the calendar21th June
Glycerol Module
Minipreps of pSB1K3-glpF8 A, B, C, D and E and digestions with EcoRI and PstI restriction enzymes
Electrophoresis
DNA precipitation and electrophoresis
Back to the calendar22th June
Glycerol Module
pSB1K3-glpF8 A, B, C, D and E digestions
Inocula of pMRB151 A, B and C
Geneclean of pSB1K3-glpF8 A-E. Purify the band that corresponds to glpF
Back to the calendar23th June
Glycerol Module
Minipreps of pMRB151 A, B and C
glpF8 geneclean
pMRB172 (mini-Tn7-nahR-Psal) obtaining
Back to the calendar24th June
Glycerol Module
pMRB151 and pMRB172 digestions
Electrophoresis and geneclean of pMRB151 A, B and C and pMRB172
Repeat digestion of pMRB151
Back to the calendar27th June
Glycerol Module
Electrophoresis of the genecleans of pMRB151 and ppMRB172
Ligations of glpF to pMRB172 and pMRB151
Back to the calendar29th June
Glycerol Module
There are 32 colonies of pMRB172-glpF and 4 colonies of pMRB151-glpF. PCR of all the colonies
Back to the calendar30th June
Glycerol Module
Electrophoresis of the PCRs
Inocula of the supposed right clones (pMRB151 1 and 2; pMRB172 6 and 8)
Back to the calendarJULY
1st July
Glycerol Module
Freeze all bacteria (for storage)
Minipreps of the rest of inocula
Electrophoresis of minipreps
Diagnostic digestions of the plasmids
Back to the calendar4nd July
Glycerol Module
Electrophoresis of all 4 digestions. PERFECT!
Measurement of the concentration using nanodrop for electroporation
Back to the calendar5th July
Glycerol Module
Obtaining of Pseudomonas putida KT2442 and inoculate it in LB medium
Back to the calendar6th July
Glycerol Module
Electroporation of pMRB172-glpF and pMRB151-glpF to P. putida KT2442
Back to the calendar7th July
Glycerol Module
PCR of the colonies obtained in the electroporation and electrophoresis
4 Inocula of the positive clones, 2 of each electroporation
Back to the calendar11th July
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar12th July
Glycerol Module
3 inocula of KT2442 with pMRB172-glpF and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source
Back to the calendar13th July
Glycerol Module
Experiment 1: Preparation of the plaques and fluorometer starting
Back to the calendar14th July
Glycerol Module
Measurement of experiment 1
Experiment 2: add octanoate and let bacteria grow till the next day
Back to the calendar18th July
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar19th July
Glycerol Module
3 inocula of KT2442 with pMRB172-glpF and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source
Back to the calendar20th July
Glycerol Module
Experiment 3: Preparation of the plaques and fluorometer starting
Back to the calendar25th July
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar26th July
Glycerol Module
3 inocula of KT2442 with pMRB172-glpF and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source
Back to the calendar27th July
Glycerol Module
Experiment 4: Preparation of the plaques and fluorometer starting
Back to the calendarAUGUST
1st August
Biofilm Module
Plate KT2442/lapG-/ΔbifA-Tn7/T133
Plate pMRB1, 166, 167, 168, 169, KT2442-T170/T159 and pRK2013 (plasmid helper for triparental mating)
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar2nd August
Biofilm Module
Inocula of 166, 167, 168, 169, KT2442-T170/T159 and pRK2013
Glycerol Module
3 inocula of KT2442 with pMRB172-glpF and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source
Back to the calendar3th August
Biofilm Module
Inocula of KT2442/lapG--Tn7/T133
Triparental mating of KT2442-T170 and KT2442-T159 with pMRB1, 166, 167, 168 and 169
Glycerol Module
Experiment 5: Preparation of the plaques and fluorometer starting
Back to the calendar4th August
Biofilm Module
Dilutions and prepare the curves of KT2442/lapG--Tn7/T133 for 20 h
Spread the plates of triparental mating onto plates with Cb (Carbamicilin) and Gm (Gentamicin)
Glycerol Module
Measurement of Experiment 5
Back to the calendar8th August
Biofilm Module
Inocula of KT2442/lapG--Tn7/T133
Segregate the plates of triparental mating
Glycerol Module
Digestion of pMRB165 (mini-Tn7-nahR-Psal-pleD*) with SpeI and PstI restriction enzymes
Digestion of pSB1K3-glpF with XbaI and PstI restriction enzymes
Back to the calendar9th August
Biofilm Module
Dilutions and prepare the curves of KT2442/lapG--T133 for 20 h
Inocula of KT2442/lapG--Tn7/T133
Inocula of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169
Glycerol Module
Electrophoresis and geneclean of the digestions of pMRB165 and pSB1K3-glpF
Electrophoresis of the purified bands
Repeat digestion of pSB1K3-glpF
Back to the calendar10th August
Biofilm Module
Dye and measure the plates of KT2442/lapG--Tn7/T133
Dilutions and prepare the curves of KT2442/lapG--T133 for 20 h
Storage KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 at -80ºC
Glycerol Module
Electrophoresis and geneclean of pSB1K3 to purify glpF
Electrophoresis of the geneclean
Back to the calendar15th August
Biofilm Module
Inocula of ΔbifA-Tn7/T128
Glycerol Module
Ligation of glpF in pMRB165
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar16th August
Biofilm Module
Dilutions and prepare the curves of ΔbifA-Tn7/T128 for 20 h
Glycerol Module
Transformation of the ligation
3 inocula of KT2442 with pMRB172-glpF and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source
Back to the calendar17th August
Biofilm Module
Dye and measure the plates of ΔbifA-Tn7/T128
Glycerol Module
Experiment 6: Preparation of the plaques and fluorometer starting
5 inocula of the colonies obtained after the transformation of the ligation
Back to the calendar18th August
Glycerol Module
Measurement of Experiment 6
Minipreps of pMRB165-glpF and freeze the rest of the inocula
Back to the calendar19th August
Biofilm Module
Plate KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 for beta-galactosidase assay
Glycerol Module
Digestions of pMRB165-glpF with XbaI and PstI restriction enzymes
Electrophoresis of the digestions. It is OK!
Back to the calendar22th August
Biofilm Module
Inocula of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar23th August
Biofilm Module
Dilutions of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 for beta-galactosidase assay
Glycerol Module
1 inocula of KT2442 with pMRB172-glpF and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source
Back to the calendar24th August
Biofilm Module
Beta-galactosidase assay (the strains with pMRB166 have not worked, it is the original Pm)
Inocula of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169
Glycerol Module
Dilutions and preparation of the microtiter plates
Back to the calendar25th August
Biofilm Module
Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay
Glycerol Module
Experiment 7: Measurement of planktonic cells and biofilms
Back to the calendar26th August
Biofilm Module
Beta-galactosidase assay
Segregate the plates of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169
Back to the calendar29th August
Biofilm Module
Inocula of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar30th August
Biofilm Module
Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay
Glycerol Module
1 inocula of KT2442 with pMRB172-glpF and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source
Back to the calendar31th August
Biofilm Module
Beta-galactosidase assay
Inocula of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169
Glycerol Module
Dilutions and preparation of the microtiter plates
Back to the calendarSEPTEMBER
1st September
Biofilm Module
Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay
Glycerol Module
Experiment 8: Measurement of planktonic cells and biofilms
Back to the calendar5th September
Biofilm Module
Inocula of KT2442-Tn7/128
Glycerol Module
Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø
Back to the calendar6th September
Biofilm Module
Dilutions and prepare the INSTANT curves of KT2442-Tn7/128 for 30 h
Glycerol Module
1 inocula of KT2442 with pMRB172-glpF and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source
Back to the calendar7th September
Biofilm Module
Dye and measure the plates of KT2442-Tn7/128
Glycerol Module
Dilutions and preparation of the microtiter plates
Back to the calendar8th September
Glycerol Module
Experiment 9: Measurement of planktonic cells and biofilms
Back to the calendar