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Revision as of 09:38, 11 October 2016
JANUARY
Monday | Tuesday | Wednesday | Thursday | Friday |
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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 |
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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 |
JANUARY
6th January
Prepare plates with LB+Km and plate the strain with pSB1K3 (E. coli DH5α)
Back to the calendar8th January
Minipreps of pSB1K3
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
Put one colony of each strain in 3 mL of LB+specific antibiotic and incubate overnight
Back to the calendar13th January
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
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
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
Ligation the vector with each PCR product
Put one colony of pMPO1035 in 3 mL of LB+Cm (Chloramphenicol)
Back to the calendar19th January
Transformation of competent cells, E. coli DH5α, with the ligations
Miniprep and measure the concentration of pMPO1035
Back to the calendar20th January
Inocula of each transformation event in 3 mL of LB+Km
PCR of pMPO1035 Back to the calendar21th January
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
Transformation of competent cells, E. coli DH5α, with the ligation
Back to the calendar25th January
Repeat the ligation of xylS2 with the vector, because the plates were contaminated
Back to the calendar26th January
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
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
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
Electrophoresis of the digestions and cut a slice that contains the fragment of interest and purify
Back to the calendarFEBRUARY
1st February
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
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
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
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
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
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
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
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
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
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
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
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
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
Inocula of each transformation in LB+Km
Transformation with the ligations
Back to the calendar19th February
Miniprep of pleD* and complex devices
Storage pMRB145, pMRB129 and pMRB130 at -80ºC
Back to the calendar22th February
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
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
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
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
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
Back to the calendar29th February
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
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
Back to the calendar2nd March
Miniprep of the culture
Segregate the plates of the transformations
Digest and electrophoresis of 124+127
Ligation Tn7 with 125 (i) and 126 (i)
Back to the calendar3th March
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)
Back to the calendar4th March
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)
Back to the calendar7th March
Inocula of 145 in 5 mL of LB
Inocula of pMRB1,124+120 and 124+127 in 3 mL of LB Back to the calendar
8th March
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
Back to the calendar9th March
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
Back to the calendar10th March
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)
Back to the calendar11th March
Transformation with the ligations
Plate the strain with 128
Digest of pMRB125 and pMRB126 as insert
Back to the calendar14th March
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)
Back to the calendar15th March
Miniprep of the cultures and 128 digest as vector
Diagnostic digestion of the Pm constructions
Storage pMRB146 at -80ºC
Transformation with the ligations
Back to the calendar16th March
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
Back to the calendar17th March
Miniprep of the cultures
Diagnostic digestion of 124+120 and Tn7+127
Transformation with the ligations
Back to the calendar18th March
The transformations with Tn7 devices didn't work, so plate Tn7 strain again
Transformation with 145+127. Now, we name this construction as pMRB147
Back to the calendar21th March
Inocula of Tn7, 147, 145+120 and 145+127
Repeat the ligation of pMRB1 (v) with 137 (i)
Back to the calendar22th March
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
Inocula of pMRB1+137
Transformation with Tn7+pMRB125
Electrophoresis of the diagnostic digestion of 145+120
Back to the calendar24th March
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
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
Inocula of Tn7+127. Now, we name this construction as pMRB151
The transformations did not go well
Back to the calendar29th March
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
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
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
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
Back to the calendar5th April
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>
Back to the calendar6th April
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
Back to the calendar7th April
Inocula of pTNS2 and 136
Ligation of Tn7 with 126 and 135
Colony PCR of the electroporations
Back to the calendar8th April
Minipreps of pTNS2 and 136
Transformation of the ligations
Electrophoresis of the colony PCR
Back to the calendar11th April
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)
Back to the calendar12th April
Minipreps and diagnostic digestion of the cultures
Transformation of the ligations
Back to the calendar13th April
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)
Back to the calendar14th April
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
Back to the calendar15th April
Measure the concentration of 136 and 134
Diagnostic digestions of 1+138, Tn7+126 and Tn7+135
Plate the strain with pTNS2
Back to the calendar18th April
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
Back to the calendar19th April
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
Back to the calendar20th April
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
Back to the calendar21th April
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
Back to the calendar22th April
Digest 152 as insert
Electroporation of KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165 with pCdrA
Back to the calendar25th April
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
Back to the calendar26th April
Digest pSB1K3 as vector
Inocula of KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165
Fluorimeter
Back to the calendar27th April
INSTANT curves of growth and biofilm of KT2442-Tn7/T134/T136/T164/T165
Failed fluorimeter
Back to the calendar28th April
Dye and measure the plates of INSTANT curves
Digest 120 as insert
Ligation Tn7+146 and Tn7+152
Back to the calendar29th April
Transformation of the ligations
Plate 128, 133, KT2442-Tn7/T136/T165 and KT2442, KT2442 lapG- and KT2442 ΔbifA
Back to the calendarMAY
2st May
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
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
Back to the calendar4th May
Inocula for curves and fluorimeter
Electrophoresis of the diagnostic digestions
Dilutions of fluorimeter and curves of KTs
Back to the calendar9th May
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
Back to the calendar10th May
Minipreps and diagnostic digestion of Tn7+120
Dilutions of yhjH and put the curves for 20 h
Segregate KT2442 lapG--T128/T133
Back to the calendar11th May
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
Back to the calendar12th May
Minipreps of the cultures
Storage pMRB159 and pMRB170 at -80ºC
Inocula of Tn7+120
Back to the calendar13th May
PCR of pMRB1 to get the gene gfp
Electrophoresis of the colony PCRs
Segregate Tn7+120
Back to the calendar16th May
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
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
Back to the calendar18th May
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
Back to the calendar19th May
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
Back to the calendar20th May
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
Back to the calendar23th May
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
Back to the calendar24th May
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
Back to the calendar25th May
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
Back to the calendar26th May
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
Back to the calendar27th May
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
Back to the calendar30th May
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 >
Back to the calendar31th May
Inocula of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Plate pMRB133
Back to the calendarJUNE
1st June
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
Back to the calendar2nd June
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
Back to the calendar3th June
Dye and measure the plates of KT2442-Tn7/T128 and lapG--Tn7/T128
Miniprep and measure the concentration of 133
Back to the calendar6th June
Plate KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Diagnostic digestion and electrophoresis of 133
Back to the calendar7th June
Inocula of KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Electroporation of KT2442, KT2442 lapG- and ΔbifA with pMRB133
Back to the calendar8th June
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
Back to the calendar9th June
Dye and measure the plates of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG--Tn7/T128
Back to the calendar