Team:UPO-Sevilla/Notebook
JANUARY
| 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 |
APRIL
| 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 |
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 |
AUGUST
| Monday | Tuesday | Wednesday | Thursday | Friday |
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 |
| 8 | 9 | 10 | 11 | 12 |
| 15 | 16 | 17 | 18 | 19 |
| 22 | 23 | 24 | 25 | 26 |
| 29 | 30 | 31 |
SEPTEMBER
| Monday | Tuesday | Wednesday | Thursday | Friday |
|---|---|---|---|---|
| 1 | 2 | |||
| 5 | 6 | 7 | 8 | 9 |
| 12 | 13 | 14 | 15 | 16 |
| 19 | 20 | 21 | 22 | 23 |
| 26 | 27 | 28 | 29 | 30 |
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 calendar21st 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 calendar22nd 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 calendar3rd 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
Miniprep 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 calendar22nd 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 calendar23rd 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.
Propionate Module
Designed and ordered primers for the PCR of the three genes from the propionate operon (scpABC).
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
Propionate Module
Transformation of the pSB1K3 plamid into E. coli DH5ɑ.
Purification of the E.coli K12 genome in order to use it as a template for the PCRs.
Back to the calendar3rd 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
Propionate Module
A colony from the transformation was picked to start a liquid culture.
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
Propionate Module
Miniprep and purification of the psB1K3 plasmid. Digestion with iGEM enzymes and electrophoresis to check that it is the right plasmid.
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
Propionate Module
The primers for the propionate operon have arrived. Started with PCR reactions for scpABC.
Digestion (XbaI, PstI) and band purification of pSB1K3.
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
Propionate Module
The PCRs of scpABC were performed to find the optimal conditions.
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
Propionate Module
The PCRs of scpABC were performed to find the optimal conditions.
The scpA fragment was obtained (Annealing temp. 43ºC).
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
Propionate Module
The PCRs of scpBC were performed to find the optimal conditions.
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
Propionate Module
The PCRs of scABC were performed to find the optimal conditions.
The scpC fragment was obtained (Annealing temp. 54ºC). Digestion of the scpA and scpC fragments with XbaI and PstI (overnight).
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
Propionate Module
Electrophoresis and purification of the desired bands from both digestions of scpA and scpC.
The PCR for the scpB gene was performed to find the optimal conditions.
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
Propionate Module
Ligation of both scpA and scpC fragments with the digested pSB1K3 plasmid (overnight). The proportions (Plasmid 1:3 Gene) were calculated using the Nanodrop.
The PCR for the scpB gene was performed to find the optimal conditions.
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
Propionate Module
The ligation mixtures were transformed into E. coli DH5ɑ.
The scpB fragment was obtained (Annealing temp. 49 ºC). It was digested overnight using XbaI and PstI.
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
Propionate Module
Electrophoresis and purification of the digested scpB fragment.
There are no colonies growing after the transformation of the ligation reaction mixture, the cloning process was unsuccessful.
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 calendar21st March
Biofilm Module
Inocula of Tn7, 147, 145+120 and 145+127
Repeat the ligation of pMRB1 (v) with 137 (i)
Back to the calendar22nd 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 calendar23rd 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
Propionate Module
The ligation reaction was performed overnight again for scpAC and also for scpB, using new proportions this time. The proportions (Plasmid 1:6 Gene) were calculated using the Nanodrop.
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
Propionate Module
Transformation of the ligation into E. coli DH5ɑ.
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!
Propionate Module
There are no colonies again for any of the genes. The cloning was unsuccessful.
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
Propionate Module
The PCRs are performed again (5x50µL PCR tubes for each gene) to obtain much more amount of DNA.
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
Propionate Module
Electrophoresis of the PCR mixture and purification of the desired bands.
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
Propionate Module
Digestion with PstI and XbaI of the scpABC fragments (overnight).
New liquid culture of E. coli DH5ɑ with the pSB1K3 plasmid.
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
Propionate Module
Electrophoresis of the digested fragments, purification of the desired bands.
Miniprep of the pSB1K3 plasmid, digestion with XbaI and PstI (overnight).
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
Propionate Module
Electrophoresis and purification of the desired band of pSB1K3.
Ligation reaction with the three scp genes, this time with the 1:6 proportions (overnight).
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
Propionate Module
Transformation of the ligation mixture into E. coli DH5ɑ.
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
Propionate Module
Two colonies are obtained for scpA and one for scpC.
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
Propionate Module
Liquid cultures are established from the colonies.
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)
Propionate Module
Miniprep from the liquid cultures. Digestion with XbaI and PstI in order to check the plasmids. The results are negative.
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
Propionate Module
New primers are designed and ordered in order to amplify the whole propionate operon. A mutagenic PCR is required (two pairs of primers needed).
Back to the calendar21st 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 calendar22nd 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
Propionate Module
The primers for the operon PCR are received. The two mutagenic PCRs (A and B) are performed to find the optimal conditions.
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)
Propionate Module
Electrophoresis in order to check the PCR results. The temperature must be increased. The PCRs are performed again. Positive results for fragment A (69ºC).
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)
Propionate Module
The PCR of the fragment B is performed again.
Electrophoresis of fragment A, purification of the desired band.
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
Propionate Module
PCR of fragment B successful (annealing temp. 68ºC). Electrophoresis and purification of the band. Digestion overnight using XbaI and PstI.
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
Propionate Module
The epi gene is ordered to a gene synthesis company.
Back to the calendarMAY
2nd 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 calendar3rd 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
Propionate Module
Measured concentration of the digested A and B fragments using NanoDrop. Both are combined for the final PCR. Optimal conditions must be obtained.
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
Propionate Module
PCR unsuccessful. The temperature is decreased.
The result is negative again and a temperature gradient is used.
Transformation of more pSB1K3 plasmid into E. coli DH5ɑ.
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
Propionate Module
A mistake is detected in the primers that were been used (the overlapping sequence is too short). New primers are designed and ordered.
Liquid cultures of the colonies obtained from the transformation of pSB1K3.
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
Propionate Module
Miniprep of the pSB1K3 plasmid.
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)
Propionate Module
The new primers are used for the PCRs of both fragments. The optimal conditions must be obtained.
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
Propionate Module
Temperature gradient PCR. The optimal temperature for fragment A is found (69ºC). The temperature for fragment B must be increased.
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
Propionate Module
Fragment B is obtained (annealing temperature 70ºC). Both fragments are purified by means of electrophoresis and band purification.
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!
Propionate Module
A PCR is performed with equal amounts of each fragment. The result is negative. A temperature gradient is performed but the results are again negative.
Back to the calendar13th May
Biofilm Module
PCR of pMRB1 to get the gene gfp
Electrophoresis of the colony PCRs
Segregate Tn7+120
Propionate Module
A PCR without primers is performed at 72ºC using high amounts of both fragments. A bit of DNA from the operon is obtained.
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
Propionate Module
The band corresponding to the whole mutated operon is purified using silica beads, but the yield is too low and the DNA is lost.
The PCR is performed again.
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
Propionate Module
Negative results for the PCR although same conditions have been used.
A concentration gradient for each of the fragments is used for a new PCR.
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
Propionate Module
Negative result for the PCR. New conditions for temperature (69ºC) and concentration of the fragments A and B are tested (without primers), and some amount of DNA corresponding to the operon is obtained.
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
Propionate Module
More PCR tubes are used in order to get more DNA. Electrophoresis and purification of the band corresponding to the operon.
This DNA is digested using XbaI and PstI overnight.
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!
Propionate Module
Electrophoresis of the digested fragments, purification of the desired band.
Back to the calendar23rd 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
Propionate Module
The epi gene has been received from the gene synthesis company. The plasmid (pUC57) is transformed into E. coli DH5ɑ.
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
Propionate Module
Many colonies are obtained for yesterday’s transformation, liquid cultures are established.
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
Propionate Module
Miniprep of the pUC57 plasmid. Digestion with EcoRI and SpeI overnight.
A stock of the pSB1K3 plasmid is digested with EcoRI and SpeI overnight.
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
Propionate Module
Electrophoresis and purification of the desired band (epi gene from pUC57 and the backbone from pSB1K3).
Ligation of both fragments, overnight using the proportions vector 1:3 gene.
Ligation of the operon fragment with the digested pSB1K3 backbone.
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)
Propionate Module
Transformation of the ligation mix for the epi gene and the operon. The plates are left at room temperature for the whole weekend.
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
Propionate Module
Seven colonies are obtained from the transformation of the ligation of epi. Liquid cultures are established from all of them.
The ligation of the operon gives negative results.
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
Propionate Module
Miniprep from the liquid cultures. Digestion with EcoRI and PstI: all the clones are positive for epi. However, due to restriction sites issues there is a fragment of the pUC57 vector cloned next to the gene. A new ligation must be done with the DNA from the pSB1K3-epi vector.
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)
Propionate Module
The clone number 1 of the pSB1K3-epi plasmid was digested overnight with XbaI and PstI in order to obtain the gene fragment.
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
Propionate Module
Electrophoresis and purification of the band corresponding to the epi gene. A new ligation reaction is set with the digested pSB1K3 backbone (overnight).
Back to the calendar3rd 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!
Propionate Module
Transformation of the ligation mix into E. coli DH5ɑ. The plates are left at room temperature for the whole weekend.
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
Propionate Module
Six colonies are obtained, and liquid cultured are prepared for all colonies.
A new PCR of the operon is performed in order to obtain more DNA (fragments A and B).
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
Propionate Module
Miniprep from the liquid cultures. Digestion with XbaI and PstI: all the clones are positive for epi. The construct is finally ready.
Electrophoresis of fragments A and B of the operon and purification of the desired bands.
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
Propionate Module
The new pSB1K3 (clone number 1) vector is digested with XbaI and PstI (overnight).
The plasmids with the expression system (nahR-Psal) and miniTn7, pMRB172 and pMRB151, are digested overnight with PstI and SpeI.
PCR of the operon using the same conditions as in the last time (same temperature and concentration of fragments A and B).
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
Propionate Module
Electrophoresis and purification of the desired bands (the epi gene and the two vector backbones from pMRB172 and pMRB151. The band corresponding to the operon from the PCR is also purified.
Two ligations reactions are set overnight (with controls) for the epi gene and each vector backbone.
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
Propionate Module
The ligation mixtures are transformed into into E. coli DH5ɑ.
Back to the calendar14th June
Glycerol Module
3 inocula of pMRB151 (A, B and C)
Propionate Module
Many colonies are obtained for each ligation. Six colonies are obtained for pMRB172 and one for pMRB151.
Liquid cultures are set for each colony.
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
Propionate Module
Miniprep from the liquid cultures. Digestion of the plasmids using XbaI and PstI. The resulting bands correspond to the size of the epi gene + the expression module. Therefore they are positive.
Back to the calendar16th June
Glycerol Module
Electrophoresis of all digestions (again)
Propionate Module
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)
Propionate Module
The clone number 1 of the pSB1K3-epi plasmid was digested overnight with EcoRI and PstI in order to obtain the gene fragment.
Transformation of the pSB1C3 plasmid into E. coli DH5ɑ.
Back to the calendar21st 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
Propionate Module
Electrophoresis and purification of the band corresponding to the epi gene.
Liquid cultures from the colonies from the transformation of pSB1C3.
Back to the calendar22nd 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
Propionate Module
Miniprep of the pSB1C3 plasmid. Digestion overnight using EcoRI and PstI.
Back to the calendar23rd June
Glycerol Module
Minipreps of pMRB151 A, B and C
glpF8 geneclean
pMRB172 (mini-Tn7-nahR-Psal) obtaining
Propionate Module
Electrophoresis and purification of the desired bands (the backbone from pSB1C3).
A new ligation reaction is set with the digested pSB1C3 backbone and the epi gene (overnight).
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
Propionate Module
Transformation of the ligation mix into E. coli DH5ɑ. The plates are left at room temperature for the whole weekend.
Back to the calendar27th June
Glycerol Module
Electrophoresis of the genecleans of pMRB151 and ppMRB172
Ligations of glpF to pMRB172 and pMRB151
Propionate Module
There are no colonies growing in the plates. These are incubated at 37ºC overnight.
The blunt pJET vector is used in order to set a ligation reaction for the operon fragment (the protocol by CloneJET is used). The ligation mixture is transformed into E. coli DH5ɑ.
Back to the calendar28th June
Glycerol Module
Transformations of the ligations to bacteria
Propionate Module
Again, negative results for the ligation of epi and pSB1C3.
Many colonies are obtained for the pJET ligation. 10 colonies are used for liquid cultures.
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
Propionate Module
Miniprep of the liquid cultures.
Digestion in order to check if the operon has been cloned. Negative result.
Another 10 colonies from the ligation in pJET are used for a liquid culture.
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)
Propionate Module
Miniprep of the liquid cultures.
Digestion in order to check if the operon has been cloned. Again, negative result.
The three genes in the operon, scpABC, are ordered to IDT using the special offer, due to the impossibility to clone them by ourselves.
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 calendar4th July
Glycerol Module
Electrophoresis of all 4 digestions. PERFECT!
Measurement of the concentration using nanodrop for electroporation
5th 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
11th 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 calendar3rd 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 calendar22nd 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 calendar23rd 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
Migration to pSB1C3
Plate pMRB122, 124, 125, 126, 135, 139, 145, 147 and 152 to put inocula next day. Plate pSB1C3
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
Migration to pSB1C3
Inocula of yesterday plates to do minipreps.
Plate pMRB123, pMRB137 and glpF to put inocula next day.
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
Migration to pSB1C3
Kit minipreps of inocula of 122, 124, 125, 126, 135, 139, 145, 147, 152 & pSB1C3.
Digestion of the inserts of the plasmids with EcoRI and PstI Digestion of the plasmids with EcoRI-PstI.
Preparative electrophoresis gel.Isolation of the bands from the gel (122 did not have a fragment. The digestion will be repeated).
Ligation of all the fragments with pSB1C3.
Inocula of yesterday’s plates (123, 137, and glpF).
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
Migration to pSB1C3
Transformation of ligation of fragments of 124, 125, 126, 135, 139, 145, 147 & 152.
Digestion of 122 with EcoRI-PstI.
Preparative electrophoresis gel.Isolation of the band from 122 fragment.
Ligation of 122 with C3.
Kit minipreprs of yesterday’s inocula (123, 137, and glpF).
Digestion of the inserts of the plasmids with EcoRI-PstI.
Back to the calendar2nd September
Biofilm Module
Beta-galactosidase assay
Plate KT2442-Tn7/128
Migration to pSB1C3
Preparative electrophoresis gel for the isolation of inserts of 123, 137, and glpF.
Plate 130, 138, 127, 129 & 146.
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
Migration to pSB1C3
Inocula of the transformation of the fragments of 124, 125, 126, 135, 139, 145, 147 & 152 (four candidates each).
Ligation of framents of 123 and glpF with C3.
Ligation of fragment of 137 to pMRB1.
Inocula of plates with 130, 138, 127, 129 & 146.
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
Migration to pSB1C3
Kit minipreps of the inocula of the plates with 130, 138, 127, 129 & 146.
Digestion of plasmids 130, 138, 127, 129 &146 with EcoRI-PstI.
Preparative electrophoresis gel and asolation of the inserts 130, 138, 127, 129 & 146.
Ligation of the inserts of 130, 138, 127, 129 & 146 with C3.
Manual miniprep of the inocula of the transformation of 124, 125, 126, 135, 139, 145, 147 & 152 with C3.
Diagnostic digestion with NotI to see the insert sizes.
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
Migration to pSB1C3
Transformation of the ligations of 130, 138, 127, 129 & 146 with C3.
Electrophoresis gel of the diagnostic digestion of the transformation of 124, 125, 126, 135, 139, 145, 147 & 152 with C3.
Second diagnostic digestion of the positives (124, 135, 152 and 125 were not positives, neither it was 126).
Electrophoresis gel with the second diagnostics digestion. Positives were 139, 145, 147 (parts BBa_K1973014, BBa_K1973016, BBa_K1973011)
Back to the calendar8th September
Glycerol Module
Experiment 9: Measurement of planktonic cells and biofilms
Migration to pSB1C3
Inocula of the transformations of 130, 138, 127, 129 & 146 with C3 (four candidates each).
Transformation of parts BBa_K1973014, BBa_K1973016, BBa_K1973011.
Inocula of plates from transformations of 123 and glpF with C3 and of 137 to pMRB1 (four candidates each, twelve candidates for Pm as it had a huge religation).
Back to the calendar9th September
Migration to pSB1C3
Manual minipreps of all the inocula from yesterday.
Digestion of the inocula from the ligations with C3 with NotI to see the insert’s sizes.
Electrophoresis gel to see the result of the diagnostic digestion. 146, 127 & 130 were negatives. 123 was also negative.
Back to the calendar12th September
Glycerol Module
Take out frozen bacteria with pSB1K3-glpF8
Obtaining of pMRB138 plasmid (pSB1K3-Pm)
Migration to pSB1C3
Second diagnostic digestion for 138 & 129.
Electrophoresis gel to see the results. Both of them were positives.
Transformation of positives of 138 and 129 with C3 to freeze them and perform kit minipreps (parts BBa_K1973013 and BBa_K1973007).
Back to the calendar13th September
Glycerol Module
Put bacteria in a liquid medium and let them grow at 37°C
Migration to pSB1C3
Plate Pm-glpF
Back to the calendar14th September
Glycerol Module
Minipreps of the plasmids
Digestion of the plasmids (pSB1K3-Pm with SpeI and PstI; pSB1K3-glpF with XbaI and PstI)
Migration to pSB1C3
Inocula of Pm-glpF.
Back to the calendar15th September
Glycerol Module
Electrophoresis of the digestions
Geneclean of glpF
Geneclean of the plasmid
Migration to pSB1C3
Kit miniprep of Pm-glpF inocula.
Digestion of Pm-glpF with EcoRI-PstI to extract the insert from the plasmid.
Preparative electrophoresis gel to isolate the insert.
Back to the calendar16th September
Migration to pSB1C3
Arrival of IDT’s DNA concerning lapC and nasR.
Transformation of the DNA (leave the plates on the table for them to grow during the weekend).
Back to the calendar19th September
Glycerol Module
Ligation of pSB1K3-Pm and glpF
Obtaining of pMRB165 (Tn7-nahR-Psal-PleD*)
Migration to pSB1C3
Inocula of the transformations of IDT’s DNA from lapC and nasR, three candidates each.
Back to the calendar20th September
Glycerol Module
Transformation of the ligation
Put bacteria with pMRB165 in a liquid medium and let them grow at 37°C
Migration to pSB1C3
Miniprep from yesterday’s inoculum of lapC1, lapC2 and lapC3, and of nasR1, nasR2 and nasR3.
Diagnostic digestion of the three of them and gel electrophoresis to see the results.
Transformation of positives of lapC (lapC2) and nasR (nasR3). Plate glpF.
Back to the calendar21st September
Glycerol Module
Put colonies in a liquid medium and let them grow at 37°C overnight
Minipreps of pMRB165 and digestion with SpeI and PstI restriction enzymes
Migration to pSB1C3
lapC and nasR from the transformations.
glpF.
Back to the calendar22nd September
Glycerol Module
Minipreps of the plasmids and diagnostic digestions
Electrophoresis. Positive results!!
Freeze bacteria containing pSB1K3-Pm-glpF for storage
Electrophoresis and geneclean of the fragment of pMRB165
Migration to pSB1C3
Miniprep of lapC, nasR, and glpF inoculum.
glpF, lapC and nasR digestion with EcoRI-PstI.
Isolation of the insert from the gel.
Ligation of Pm-glpF, glpF, lapC, nasR and 125 with pSB1C3.
Back to the calendar23rd September
Migration to pSB1C3
Transformation of ligation of Pm-glpF, 125, glpF, lapC & nasR with pSB1C3 in DH5α (leave the plates on the table for them growing during the weekend).
Back to the calendar26th September
Glycerol Module
Ligation of pMRB165 and glpF
Migration to pSB1C3
Inocula of the transformations of the ligations (four candidates of each, Pm-glpF, glpF, 125, lapC & nasR). Plate pMRB120, 127, 140 & 146.
Back to the calendar27th September
Glycerol ModuleTransformation of the ligation
Migration to pSB1C3
Manual minipreps of yesterday’s inocula of Pm-glpF, glpF, 125, lapC & nasR.
Diagnostic digestion of the ligations with NotI in order to see the fragment size of Pm-glpF, glpF, 125, lapC & nasR.
Gel electrophoresis of the digestion.
Second diagnostic digestion of the positives of Pm-glpF, glpF, 125, lapC & nasR.
Gel electrophoresis of the digestion.
Transformation of positive constructs of Pm-glpF, glpF, 125, lapC & nasR to freeze them and perform kit minipreps for submitting.
Inocula of pMRB120, 127, 140 & 146.
Back to the calendar28th September
Glycerol Module
Put colonies in a liquid medium and let them grow at 37ºC overnight
Migration to pSB1C3
Inocula of yesterday’s transformations of positives of Pm-glpF, glpF, 125, lapC & nasR.
Manual miniprep of 120, 127, 140 & 146.
Digestion of 120, 127, 140 & 146 with EcoRI and PstI.
Preparative electrophoresis gel and band asolation of 120, 127, 140 & 146 insert.
Ligation of 120, 127, 140 & 146 + C3.
Ligation of 121 + C3 (already have it stored in the freezer, cut with EcoRI-PstI from previous ligations).
Back to the calendar29th September
Glycerol Module
Minipreps of the plasmids and diagnostic digestions
Electrophoresis. Positive results!!
Freeze bacteria containing Tn7-nahR-Psal-PleD*-glpF for storage
Migration to pSB1C3
Kit minipreps of yesterday’s inocula of postives of Pm-glpF, glpF, 125, lapC & nasR.
Mesuring of DNA concentraton and dilution of it until 25 ng/ul for the submitting plate (parts BBa_K1973035,BBa_K1973026,BBa_K1973003, BBa_K1973036, BBa_K1973037).
Transformation of the ligation of 120, 127, 140, 121 & 146 + C3.
Back to the calendarOCTOBER
3rd October
Migration to pSB1C3
Inocula of the transformation of the ligations of 120, 127, 140, 121 & 146 + C3.
Back to the calendar4th October
Migration to pSB1C3
Manual minipreps of yesterday’s inocula of 120, 127, 140, 121 & 146 + C3.
Diagnostic digestion of the ligations with NotI in order to see the fragment size of 120, 127, 140, 121 & 146 + C3.
Gel electrophoresis of the digestion. 146 was again negative.
Second diagnostic digestion of the positives of 120, 127, 140 &121+ C3 (let it overnight).
Back to the calendar5th October
Migration to pSB1C3
Gel electrophoresis of the digestion.
Transformation of positive constructs 120, 127, 140 & 121 + C3 to freeze them and perform kit minipreps for submitting (146 had no positives).
Back to the calendar6th October
Migration to pSB1C3
Inocula from yesterday’s transformation (constructs 120, 127, 140 &121 + C3).
Plate all miniTn7 constructs for their minipreps made and DNA dilution performed (parts BBa_K1973006, BBa_K1973009, BBa_K1973010, BBa_K1973012, BBa_K1973019, BBa_K1973021, BBa_K1973022, BBa_K1973023, BBa_K1973024, BBa_K1973025, BBa_K1973027, BBa_K1973028, BBa_K1973029, BBa_K1973031, BBa_K1973032, BBa_K1973038).
Back to the calendar7th October
Migration to pSB1C3
Kit miniprep of the inocula performed yesterday.
Measuring of DNA concentration and dilution unit 25 ng/ul (parts BBa_K1973033, BBa_K1973001, BBa_K1973015, BBa_K1973000).
Parts BBa_K1973031, BBa_K1973032 did not grow. Made a mistake when plating them.
Back to the calendar10th October
Migration to pSB1C3
Inocula of all miniTn7 devices for kit minipreps being performed on them.
Secong attempt of ligation of 123, 124, 130 and 135 with C3.
Back to the calendar11th October
Migration to pSB1C3
Kit miniprep of all the inocula made yesterday.
Measure DNA concentration of all the miniTn7 devices and dilute them to 25 ng/ul.
Transformation of the ligations performed yesterday.
Back to the calendar12th October
Migration to pSB1C3
Inocula of the transformatios performed yesterday (four candidates each construction).
Back to the calendar13th October
Migration to pSB1C3
Kit minipreps of the inocula put yesterday.
Digestion of the minipreps with NotI to see the inserts sizes. All constructs had some positive when seen in the electrophoresis gel.
Second digestion performed to confirm the construcions.
Electrophoresis gel from the positives digested. All of them were positives.
As the minipreps where from the kit, measure of the DNA concentration and dilution until 25 ng/ul.
Back to the calendar17th October
Migration to pSB1C3
Inocula of the transformation of Pm with pMRB1 (six candidates).
Inocula of 146, 152, 126 and epimerase from the transformations of the ligations of them with C3 (four candidates each).
Plate Tn7-nahR-Psal-glpF and Tn7-nahR-Psal-nasF-glpF.
Measuring of DNA concentration of parts BBa_K1973005, BBa_K1973031, BBa_K1973032, and dilution to 25 ng/ul.
Transformation of the DNA of positive from ligation of 122 with C3.
Back to the calendar18th October
Migration to pSB1C3
Kit minipreps of the inocula from the ligations.
Diagnostic digestion with NotI to see insert sizes (146, 126, 152 and epimerase).
Diagnostic digestion with EcoRI-HindIII to see if Pm ligation with pMRB1 gone well.
Electrophoresis gel to see the results of both digestions (126 and 152 were negative. We cannot send them to the registry, and the ligation with Pm also was negative).
Second diagnostic digestion with EcoRI and EcoRV of the positives from first digestion.
Electrophoresis gel to see the results.
Dilution of the positives’ DNA until 25 ng/ul (parts BBa_K1973030 and BBa_K1973017).
Inocula of Tn7-nahR-Psal-glpF, Tn7-nahR-Psal-nasF-glpF, and 122.
Back to the calendar19th October
Migration to pSB1C3
Kit miniprep of Tn7-nahR-Psal-glpF, Tn7-nahR-Psal-nasF-glpF and 122 inocula.
Measure of DNA concentration of these minipreps and dilution to 25 ng/ul.
PREPARATION OF SUBMISSION PLATE, and done the on-line form for the submissions. Certificate submission of the plate.
Back to the calendar