1^st January

4^nd January

5^th January

6^th January

Prepare plates with LB+Km and plate the strain with pSB1K3 (E. coli DH5α)

7^th January

Inocula of pSB1K3 in 15 mL of LB+Km (kanamycin)

8^th 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)

11^th January

Put one colony of each strain in 3 mL of LB+specific antibiotic and incubate overnight

12^th January

Minipreps and measure the concentration of the cultures

13^th 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

14^th 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

15^th 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)

18^th January

Ligation the vector with each PCR product

Put one colony of pMPO1035 in 3 mL of LB+Cm (Chloramphenicol)

19^th January

Transformation of competent cells, E. coli DH5α, with the ligations

Miniprep and measure the concentration of pMPO1035

20^th January

Inocula of each transformation event in 3 mL of LB+Km

21^th 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

22^th January

Transformation of competent cells, E. coli DH5α, with the ligation

25^th January

Repeat the ligation of xylS2 with the vector, because the plates were contaminated

26^th January

Send to sequence 4 constructions (lapG, nahR-Psal, nasF, yhjH)

Transformation of competent cells, E. coli DH5α, with the ligation

27^th 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

28^th 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

29^th January

Electrophoresis of the digestions and cut a slice that contains the fragment of interest and purify

1^st February

Inocula of each transformation and xylS2 in 3 mL of LB+Km

Ligation of yhjH, lapG and nasF with nahR-Psal

2^nd 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*

3^th 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

4^th 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

5^th 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

8^th 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

9^th 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

10^th 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

11^th 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

12^th 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

15^th 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

16^th 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

17^th 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)

18^th February

Inocula of each transformation in LB+Km

Transformation with the ligations

19^th February

Miniprep of pleD* and complex devices

Storage pMRB145, pMRB129 and pMRB130 at -80ºC

22^th February

Incocula of each transformation in LB

Plate the strains with pMRB124 (xylS2) and pTNS2 (helper of Tn7)

Digest pMRB129 and pMRB130 as insert

23^th 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

24^th 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

25^th 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

26^th 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

29^th 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

1^st 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

2^nd 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)

3^th 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)

4^th 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)

7^th 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

8^th 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

9^th 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

10^th 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)

11^th March

Transformation with the ligations

Plate the strain with 128

Digest of pMRB125 and pMRB126 as insert

14^th 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)

15^th March

Miniprep of the cultures and 128 digest as vector

Diagnostic digestion of the Pm constructions

Storage pMRB146 at -80ºC

Transformation with the ligations

16^th 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

17^th March

Miniprep of the cultures

Diagnostic digestion of 124+120 and Tn7+127

Transformation with the ligations

18^th 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

21^th March

Inocula of Tn7, 147, 145+120 and 145+127

Repeat the ligation of pMRB1 (v) with 137 (i)

22^th 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)

23^th March

Inocula of pMRB1+137

Transformation with Tn7+pMRB125

Electrophoresis of the diagnostic digestion of 145+120

24^th 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)

25^th 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

28^th March

Inocula of Tn7+127. Now, we name this construction as pMRB151

The transformations did not go well

29^th 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

30^th March

Transformation of the ligations

31^th March

Inocula of 1+137/138/139/140, 120+145,120+124 and Tn7+147

Segregate the transformation of Tn7+126

1^st April

Minipreps and diagnostic digestion of the 1+137/138/139/140, 120+124, 145+120 and Tn7+147

Electrophoresis of the diagnostic digestion

4^nd 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

5^th 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>

6^th 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

7^th April

Inocula of pTNS2 and 136

Ligation of Tn7 with 126 and 135

Colony PCR of the electroporations

8^th April

Minipreps of pTNS2 and 136

Transformation of the ligations

Electrophoresis of the colony PCR

11^th 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)

12^th April

Minipreps and diagnostic digestion of the cultures

Transformation of the ligations

13^th 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)

14^th 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

15^th April

Measure the concentration of 136 and 134

Diagnostic digestions of 1+138, Tn7+126 and Tn7+135

Plate the strain with pTNS2

18^th 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

19^th 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

20^th 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

21^th 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

22^th April

Digest 152 as insert

Electroporation of KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165 with pCdrA

25^th 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

26^th April

Digest pSB1K3 as vector

Inocula of KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165

Fluorimeter

27^th April

INSTANT curves of growth and biofilm of KT2442-Tn7/T134/T136/T164/T165

Failed fluorimeter

28^th April

Dye and measure the plates of INSTANT curves

Digest 120 as insert

Ligation Tn7+146 and Tn7+152

29^th April

Transformation of the ligations

Plate 128, 133, KT2442-Tn7/T136/T165 and KT2442, KT2442 lapG^- and KT2442 ΔbifA

2^st 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

3^nd 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

4^th May

Inocula for curves and fluorimeter

Electrophoresis of the diagnostic digestions

Dilutions of fluorimeter and curves of KTs

5^th May

Dye and measure the curves of pleD*

Take the data of the fluorimeter

6^th May

Plate the variants of KT2442 with yhjH

Transformation of Tn7+120

9^th 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

10^th May

Minipreps and diagnostic digestion of Tn7+120

Dilutions of yhjH and put the curves for 20 h

Segregate KT2442 lapG^--T128/T133

11^th 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

12^th May

Minipreps of the cultures

Storage pMRB159 and pMRB170 at -80ºC

Inocula of Tn7+120

13^th May

PCR of pMRB1 to get the gene gfp

Electrophoresis of the colony PCRs

Segregate Tn7+120

16^th May

Inocula of pleD* constructions in Tn7 and KT2442 lapG^--T128

Inocula of Tn7+120

Colony PCR and electrophoresis of KT2442 lapG^--T133

17^th 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

18^th 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

19^th 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

20^th 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

23^th 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

24^th 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

25^th 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

26^th 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

27^th 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

30^th 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 >

31^th May

Inocula of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG^--Tn7/T128

Plate pMRB133

1^st 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

2^nd 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

3^th June

Dye and measure the plates of KT2442-Tn7/T128 and lapG^--Tn7/T128

Miniprep and measure the concentration of 133

6^th June

Plate KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG^--Tn7/T128

Diagnostic digestion and electrophoresis of 133

7^th June

Inocula of KT2442-Tn7/T134/T164 and KT2442-Tn7/T128 and lapG^--Tn7/T128

Electroporation of KT2442, KT2442 lapG^- and ΔbifA with pMRB133

8^th 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

9^th June

Dye and measure the plates of KT2442-T134/T164 and KT2442-Tn7/T128 and lapG^--Tn7/T128

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