7^th January

Biofilm Module

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

8^th 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)

11^th 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

12^th January

Biofilm Module

Minipreps of pSB1K3

Minipreps and measure the concentration of the cultures

13^th 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

14^th 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

15^th 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)

18^th January

Biofilm Module

Ligation the vector with each PCR product

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

19^th January

Biofilm Module

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

Miniprep and measure the concentration of pMPO1035

20^th January

Biofilm Module

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

PCR of pMPO1035

21^th 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

22^th January

Biofilm Module

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

25^th January

Biofilm Module

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

26^th January

Biofilm Module

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

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

27^th 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

28^th 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

29^th January

Biofilm Module

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

1^st February

Biofilm Module

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

Ligation of yhjH, lapG and nasF with nahR-Psal

2^nd 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*

3^th 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

4^th 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

5^th 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

8^th 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

9^th 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

10^th 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

11^th 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

12^th 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

15^th 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

16^th 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

17^th 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)

18^th February

Biofilm Module

Inocula of each transformation in LB+Km

Transformation with the ligations

19^th February

Biofilm Module

Miniprep of pleD* and complex devices

Storage pMRB145, pMRB129 and pMRB130 at -80ºC

22^th 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

23^th 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

24^th 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

25^th 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

26^th 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

29^th 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

1^st 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.

2^nd 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

3^th 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

4^th 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

7^th 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

8^th 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

9^th 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

10^th 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

11^th 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

14^th 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

15^th 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

16^th 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

17^th 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

18^th 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

21^th March

Biofilm Module

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

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

22^th 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)

23^th March

Biofilm Module

Inocula of pMRB1+137

Transformation with Tn7+pMRB125

Electrophoresis of the diagnostic digestion of 145+120

24^th 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)

25^th 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

28^th March

Biofilm Module

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

The transformations did not go well

29^th 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

30^th March

Biofilm Module

Transformation of the ligations

31^th March

Biofilm Module

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

Segregate the transformation of Tn7+126

1^st 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

4^nd 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

5^th 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

6^th 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!

7^th 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

8^th 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

11^th 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

12^th 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

13^th 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

14^th 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

15^th 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

18^th 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

19^th 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)

20^th 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

21^th 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

22^th 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

25^th 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

26^th 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)

27^th 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)

28^th 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

29^th April

Biofilm Module

Transformation of the ligations

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

2^st 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

3^nd 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

4^th 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

5^th 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

6^th 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

9^th 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)

10^th 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

11^th 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

12^th 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!

13^th May

Biofilm Module

PCR of pMRB1 to get the gene gfp

Electrophoresis of the colony PCRs

Segregate Tn7+120

16^th 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

17^th 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

18^th 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

19^th 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

20^th 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!

23^th 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

24^th 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

25^th 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

26^th 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

27^th 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)

30^th 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

31^th 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

1^st 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)

2^nd 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

3^th 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!

6^th 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

7^th 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

8^th 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

9^th 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

10^th June

Biofilm Module

Storage KT2442/lapG^-/ΔbifA-T133 at -80ºC

Glycerol Module

Electrophoresis of the purified band. There is nothing!

13^th June

Glycerol Module

Spread bacteria with pMRB151on agar plates and let them grow at 37ºC

14^th June

Glycerol Module

3 inocula of pMRB151 (A, B and C)

15^th 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

16^th June

Glycerol Module

Electrophoresis of all digestions (again)

17^th June

Glycerol Module

Geneclean pSB1K3-glpF8 digested

pSB1K3-glpF8 digestion with EcoRI and XbaI

Spread bacteria with pSB1K3-glpF8 and incubate at room temperature

20^th June

Glycerol Module

Electrophoresis

5 inocula (pSB1K3-glpF8 A, B , C, D and E)

21^th 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

22^th 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

23^th June

Glycerol Module

Minipreps of pMRB151 A, B and C

glpF8 geneclean

pMRB172 (mini-Tn7-nahR-Psal) obtaining

24^th June

Glycerol Module

pMRB151 and pMRB172 digestions

Electrophoresis and geneclean of pMRB151 A, B and C and pMRB172

Repeat digestion of pMRB151

27^th June

Glycerol Module

Electrophoresis of the genecleans of pMRB151 and ppMRB172

Ligations of glpF to pMRB172 and pMRB151

28^th June

Glycerol Module

Transformations of the ligations to bacteria

29^th June

Glycerol Module

There are 32 colonies of pMRB172-glpF and 4 colonies of pMRB151-glpF. PCR of all the colonies

30^th June

Glycerol Module

Electrophoresis of the PCRs

Inocula of the supposed right clones (pMRB151 1 and 2; pMRB172 6 and 8)

1^st July

Glycerol Module

Freeze all bacteria (for storage)

Minipreps of the rest of inocula

Electrophoresis of minipreps

Diagnostic digestions of the plasmids

4^nd July

Glycerol Module

Electrophoresis of all 4 digestions. PERFECT!

Measurement of the concentration using nanodrop for electroporation

5^th July

Glycerol Module

Obtaining of Pseudomonas putida KT2442 and inoculate it in LB medium

6^th July

Glycerol Module

Electroporation of pMRB172-glpF and pMRB151-glpF to P. putida KT2442

7^th July

Glycerol Module

PCR of the colonies obtained in the electroporation and electrophoresis

4 Inocula of the positive clones, 2 of each electroporation

8^th July

Glycerol Module

Freeze all four bacteria

11^th July

Glycerol Module

Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø

12^th 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

13^th July

Glycerol Module

Experiment 1: Preparation of the plaques and fluorometer starting

14^th July

Glycerol Module

Measurement of experiment 1

Experiment 2: add octanoate and let bacteria grow till the next day

15^th July

Glycerol Module

Measurement of Experiment 2

18^th July

Glycerol Module

Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø

19^th 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

20^th July

Glycerol Module

Experiment 3: Preparation of the plaques and fluorometer starting

21^th July

Glycerol Module

Measurement of Experiment 3

22^th July

25^th July

Glycerol Module

Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø

26^th 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

27^th July

Glycerol Module

Experiment 4: Preparation of the plaques and fluorometer starting

28^th July

Glycerol Module

Measurement of Experiment 4

29^th July

1^st 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

2^nd 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

3^th 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

4^th 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

5^th August

Biofilm Module

Dye and measure the plates of KT2442/lapG^--Tn7/T133

8^th 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

9^th 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

10^th 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

11^th August

Biofilm Module

Dye and measure the plates of KT2442/lapG^--Tn7/T133

12^th August

15^th 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

16^th 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

17^th 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

18^th August

Glycerol Module

Measurement of Experiment 6

Minipreps of pMRB165-glpF and freeze the rest of the inocula

19^th 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!

22^th 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

23^th 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

24^th 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

25^th 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

26^th 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

29^th 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

30^th 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

31^th 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

1^st 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

2^nd September

Biofilm Module

Beta-galactosidase assay

Plate KT2442-Tn7/128

5^th September

Biofilm Module

Inocula of KT2442-Tn7/128

Glycerol Module

Spread in plaques KT2442 with pMRB172-glpF and KT2442 with Tn7 Ø

6^th 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

7^th September

Biofilm Module

Dye and measure the plates of KT2442-Tn7/128

Glycerol Module

Dilutions and preparation of the microtiter plates

8^th September

Glycerol Module

Experiment 9: Measurement of planktonic cells and biofilms

9^th September

12^th September

13^th September

14^th September

15^th September

16^th September

19^th September

20^th September

21^th September

22^th September

23^th September

26^th September

27^th September

28^th September

29^th September

30^th September