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.

Propionate Module

Designed and ordered primers for the PCR of the three genes from the propionate operon (scpABC).

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

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.

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

Propionate Module

A colony from the transformation was picked to start a liquid culture.

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

Propionate Module

Miniprep and purification of the psB1K3 plasmid. Digestion with iGEM enzymes and electrophoresis to check that it is the right plasmid.

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

Propionate Module

The primers for the propionate operon have arrived. Started with PCR reactions for scpABC.

Digestion (XbaI, PstI) and band purification of pSB1K3.

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

Propionate Module

The PCRs of scpABC were performed to find the optimal conditions.

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

Propionate Module

The PCRs of scpABC were performed to find the optimal conditions.

The scpA fragment was obtained (Annealing temp. 43ºC).

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

Propionate Module

The PCRs of scpBC were performed to find the optimal conditions.

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

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).

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

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.

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

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.

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

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.

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

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.

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

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.

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

Propionate Module

Transformation of the ligation into E. coli DH5ɑ.

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!

Propionate Module

There are no colonies again for any of the genes. The cloning was unsuccessful.

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

Propionate Module

The PCRs are performed again (5x50µL PCR tubes for each gene) to obtain much more amount of DNA.

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

Propionate Module

Electrophoresis of the PCR mixture and purification of the desired bands.

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

Propionate Module

Digestion with PstI and XbaI of the scpABC fragments (overnight).

New liquid culture of E. coli DH5ɑ with the pSB1K3 plasmid.

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

Propionate Module

Electrophoresis of the digested fragments, purification of the desired bands.

Miniprep of the pSB1K3 plasmid, digestion with XbaI and PstI (overnight).

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

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).

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

Propionate Module

Transformation of the ligation mixture into E. coli DH5ɑ.

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

Propionate Module

Two colonies are obtained for scpA and one for scpC.

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

Propionate Module

Liquid cultures are established from the colonies.

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)

Propionate Module

Miniprep from the liquid cultures. Digestion with XbaI and PstI in order to check the plasmids. The results are negative.

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

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).

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

Propionate Module

The primers for the operon PCR are received. The two mutagenic PCRs (A and B) are performed to find the optimal conditions.

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)

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).

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)

Propionate Module

The PCR of the fragment B is performed again.

Electrophoresis of fragment A, purification of the desired band.

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

Propionate Module

PCR of fragment B successful (annealing temp. 68ºC). Electrophoresis and purification of the band. Digestion overnight using XbaI and PstI.

29^th 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.

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

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.

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

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.

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

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.

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

Propionate Module

Miniprep of the pSB1K3 plasmid.

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)

Propionate Module

The new primers are used for the PCRs of both fragments. The optimal conditions must be obtained.

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

Propionate Module

Temperature gradient PCR. The optimal temperature for fragment A is found (69ºC). The temperature for fragment B must be increased.

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

Propionate Module

Fragment B is obtained (annealing temperature 70ºC). Both fragments are purified by means of electrophoresis and band purification.

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!

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.

13^th 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.

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

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.

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

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.

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

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.

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

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.

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!

Propionate Module

Electrophoresis of the digested fragments, purification of the desired band.

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

Propionate Module

The epi gene has been received from the gene synthesis company. The plasmid (pUC57) is transformed into E. coli DH5ɑ.

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

Propionate Module

Many colonies are obtained for yesterday’s transformation, liquid cultures are established.

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

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.

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

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.

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)

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.

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

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.

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

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.

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)

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.

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

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).

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!

Propionate Module

Transformation of the ligation mix into E. coli DH5ɑ. The plates are left at room temperature for the whole weekend.

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

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).

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

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.

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

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).

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

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.

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

Propionate Module

The ligation mixtures are transformed into into E. coli DH5ɑ.

14^th 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.

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

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.

16^th June

Glycerol Module

Electrophoresis of all digestions (again)

Propionate Module

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)

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.

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

Propionate Module

Electrophoresis and purification of the band corresponding to the epi gene.

Liquid cultures from the colonies from the transformation of pSB1C3.

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

Propionate Module

Miniprep of the pSB1C3 plasmid. Digestion overnight using EcoRI and PstI.

23^th 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).

24^th 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.

27^th 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.

28^th 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.

29^th 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.

30^th 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.

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

Migration to pSB1C3

Plate pMRB122, 124, 125, 126, 135, 139, 145, 147 and 152 to put inocula next day. Plate pSB1C3

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

Migration to pSB1C3

Inocula of yesterday plates to do minipreps.

Plate pMRB123, pMRB137 and glpF to put inocula next day.

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

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).

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

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.

2^nd 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.

5^th 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.

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

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.

7^th 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)

8^th 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).

9^th 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.

12^th 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).

13^th September

Glycerol Module

Put bacteria in a liquid medium and let them grow at 37°C

Migration to pSB1C3

Plate Pm-glpF

14^th 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.

15^th 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.

16^th 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).

19^th 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.

20^th 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.

21^th 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.

22^th 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.

23^th 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).

26^th 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.

27^th September

Transformation 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.

28^th 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).

29^th 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.

30^th September