iGEM 2016 — Team UPO-Sevilla

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# Team:UPO-Sevilla/Notebook

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  + [Engagement](https://2016.igem.org/Team%3AUPO-Sevilla/Engagement)

# NOTEBOOK

**JANUARY**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  |  |  |  | [1](#11) |
| [4](#14) | [5](#15) | [6](#16) | [7](#17) | [8](#18) |
| [11](#111) | [12](#112) | [13](#113) | [14](#114) | [15](#115) |
| [18](#118) | [19](#119) | [20](#120) | [21](#121) | [22](#122) |
| [25](#125) | [26](#126) | [27](#127) | [28](#128) | [29](#129) |

**FEBRUARY**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
| [1](#21) | [2](#22) | [3](#23) | [4](#24) | [5](#25) |
| [8](#28) | [9](#29) | [10](#210) | [11](#211) | [12](#212) |
| [15](#215) | [16](#216) | [17](#217) | [18](#218) | [19](#219) |
| [22](#222) | [23](#223) | [24](#224) | [25](#225) | [26](#226) |
| [29](#229) |

**MARCH**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  | [1](#31) | [2](#32) | [3](#33) | [4](#34) |
| [7](#37) | [8](#38) | [9](#39) | [10](#310) | [11](#311) |
| [14](#314) | [15](#315) | [16](#316) | [17](#317) | [18](#318) |
| [21](#321) | [22](#322) | [23](#323) | [24](#324) | [25](#325) |
| [28](#328) | [29](#329) | [30](#330) | [31](#331) |

**APRIL**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  |  |  |  | [1](#41) |
| [4](#44) | [5](#45) | [6](#46) | [7](#47) | [8](#48) |
| [11](#411) | [12](#412) | [13](#413) | [14](#414) | [15](#415) |
| [18](#418) | [19](#419) | [20](#420) | [21](#421) | [22](#422) |
| [25](#425) | [26](#426) | [27](#427) | [28](#428) | [29](#429) |

**MAY**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
| [2](#52) | [3](#53) | [4](#54) | [5](#55) | [6](#56) |
| [9](#59) | [10](#510) | [11](#511) | [12](#512) | [13](#513) |
| [16](#516) | [17](#517) | [18](#518) | [19](#519) | [20](#520) |
| [23](#523) | [24](#524) | [25](#525) | [26](#526) | [27](#527) |
| [30](#530) | [31](#5) |

**JUNE**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  |  | [1](#61) | [2](#62) | [3](#63) |
| [6](#66) | [7](#67) | [8](#68) | [9](#69) | [10](#610) |
| [13](#613) | [14](#614) | [15](#615) | [16](#616) | [17](#617) |
| [20](#620) | [21](#621) | [22](#622) | [23](#623) | [24](#624) |
| [27](#627) | [28](#628) | [29](#629) | [30](#630) |

**JULY**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  |  |  |  | [1](#71) |
| [4](#74) | [5](#75) | [6](#76) | [7](#77) | [8](#78) |
| [11](#712) | [12](#712) | [13](#713) | [14](#714) | [15](#715) |
| [18](#718) | [19](#719) | [20](#720) | [21](#721) | [22](#722) |
| [25](#725) | [26](#726) | [27](#727) | [28](#728) | [29](#729) |

**AUGUST**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
| [1](#81) | [2](#82) | [3](#83) | [4](#84) | [5](#85) |
| [8](#88) | [9](#89) | [10](#810) | [11](#811) | [12](#812) |
| [15](#815) | [16](#816) | [17](#817) | [18](#818) | [19](#819) |
| [22](#822) | [23](#823) | [24](#824) | [25](#825) | [26](#826) |
| [29](#829) | [30](#830) | [31](#831) |

**SEPTEMBER**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
|  |  |  | [1](#91) | [2](#92) |
| [5](#95) | [6](#96) | [7](#97) | [8](#98) | [9](#99) |
| [12](#912) | [13](#913) | [14](#914) | [15](#915) | [16](#916) |
| [19](#919) | [20](#920) | [21](#921) | [22](#922) | [23](#923) |
| [26](#926) | [27](#927) | [28](#928) | [29](#929) | [30](#930) |

**OCTOBER**

| Monday | Tuesday | Wednesday | Thursday | Friday |
| --- | --- | --- | --- | --- |
| [3](#103) | [4](#104) | [5](#105) | [6](#106) | [7](#107) |
| [10](#1010) | [11](#1011) | [12](#1012) | [13](#1013) | [14](#1014) |
| [17](#1017) | [18](#1018) | [19](#1019) | [20](#1020) | [21](#1021) |
| 24 | 25 | 26 | 27 | 28 |
| 31 |

## **JANUARY**

### **7th January**

**Biofilm Module**

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

[Back to the calendar](#calendar)

### **8th January**

**Biofilm Module**

Plate the strains of *E. coli* DH5α with pMPO364 (*nasF* and *nahR-*P*sal*), JB3Tc19::PleD (*pleD\**), pMPO52 (P*m*) and pMRB11(*yhjH*), and *P. putida* KT2442 (*lapG*)

[Back to the calendar](#calendar)

### **11th 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

**Starting the metabolic pathway software tool.**

[Back to the calendar](#calendar)

### **12th January**

**Biofilm Module**

Minipreps of pSB1K3

Minipreps and measure the concentration of the cultures

[Back to the calendar](#calendar)

### **13th January**

**Biofilm Module**

14 first primers have arrived. Resuspend them, dilute until 10 μM and storage at -20ºC

PCR of pMPO364 and pMRB11 [45ºC]

PCR of pJBeTc19::pleD and *Pseudomonas putida* KT2442 chromosome [60ºC]

Electrophoresis of both PCR

[Back to the calendar](#calendar)

### **14th January**

**Biofilm Module**

Repeat the PCR at 45ºC and at 60ºC

Electrophoresis of both PCR

Purify the 5 PCR products (*lapG, nahR-*P*sal, nasF, pleD\*, yhjH*) and store them at -20ºC

[Back to the calendar](#calendar)

### **15th January**

**Biofilm Module**

Digest the purified PCR products and pSB1K3 where we will clone the PCR products

Electrophoresis of the digested PCR products and vector and cut a slice of the gel that contain the fragment of interest and purify

Plate the strain with pMPO1035 (*xylS2*)

[Back to the calendar](#calendar)

### **18th January**

**Design of the microscopic simulation of biofilm growth model.**

**Biofilm Module**

Ligation the vector with each PCR product

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

[Back to the calendar](#calendar)

### **19th January**

**Biofilm Module**

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

Miniprep and measure the concentration of pMPO1035

[Back to the calendar](#calendar)

### **20th January**

**Biofilm Module**

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

PCR of pMPO1035

[Back to the calendar](#calendar)

### **21st January**

**Biofilm Module**

Miniprep the plasmid DNA of the transformations

Diagnostic digest the plasmids with PstI and XbaI

Electrophoresis of the digestions and the PCR. All the fragments had the correct length

Purify the PCR of *xylS2* and digest with PstI and XbaI

Purify the digestion of *xylS2* and ligation with pSB1K3

[Back to the calendar](#calendar)

### **22nd January**

**Biofilm Module**

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

[Back to the calendar](#calendar)

### **25th January**

**Biofilm Module**

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

**Metabolic pathway software tool was eventually developed.**

[Back to the calendar](#calendar)

### **26th January**

**Biofilm Module**

Send to sequence 4 constructions (*lapG, nahR-*P*sal, nasF, yhjH*)

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

[Back to the calendar](#calendar)

### **27th January**

**Biofilm Module**

The transformation was unsuccessful, so we did a electrophoresis of the vector and the insert (xylS2) to test that we have DNA in the sample

Repeat the ligation of xylS2 with the vector

Dilute until 10 μM the primers that we use to do a overlap extension PCR

[Back to the calendar](#calendar)

### **28th January**

**Biofilm Module**

Test the received sequences of *lapG, nahR-*P*sal, 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-*P*sal*), pMRB121 (*lapG*), pMRB122 (*yhjH*) and pMRB123 (*nasF*)

Digest pMRB121, pMRB122 and pMRB123 as vector and pMRB120 as insert

[Back to the calendar](#calendar)

### **29th January**

**Biofilm Module**

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

[Back to the calendar](#calendar)

## **FEBRUARY**

### **1st February**

**The kinetics population model was designed.**

**Biofilm Module**

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

Ligation of *yhjH, lapG* and *nasF* with *nahR-*P*sal*

[Back to the calendar](#calendar)

### **2nd February**

**Biofilm Module**

Miniprep and diagnostic digestion of xylS2

Storage the pMRB120, pMRB121, pMRB122 and pMRB123 strains at -80ºC

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

Send to sequence the constructions of xylS2 and pleD\*

[Back to the calendar](#calendar)

### **3rd 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 P*m*

[Back to the calendar](#calendar)

### **4th February**

**Biofilm Module**

Inocula of the transformation in 3 mL of LB+Km

Electrophoresis of PCR of *P*m, but it did not go well

Repeat the PCR of *P*m

[Back to the calendar](#calendar)

### **5th February**

**Biofilm Module**

Miniprep of *nahR-*P*sal-lapG, nahR-*P*sal-yhjH* and *nahR-*P*sal-nasF*

Diagnostic digest of these plasmids

Test the received sequences of *xylS2* and *pleD\**. Only *xylS2* is correct, so do a transformation to introduce the plasmid, now we name this construction as pMRB124

[Back to the calendar](#calendar)

### **8th 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 P*m* RM 1 (the fragment of P*m* amplified with the primer reverse and mutagenic forward 1) did not go well

[Back to the calendar](#calendar)

### **9th February**

**Biofilm Module**

Inocula of each transformation in LB+Km

Storage the pMRB124 strain at -80ºC

Repeat the PCR of P*m* RM 1

Electrophoresis of the PCR

[Back to the calendar](#calendar)

### **10th February**

**Biofilm Module**

Storage the pMRB125, pRMB126 and pMRB127 strains at -80ºC

Purify all the PCR products of P*m*

Digest pMRB127 with EcoRI and SpeI as insert

Ligation of the vector with *pleD\**, and *lapG* and *yhjH* with *nahR-*P*sal-nasF*

[Back to the calendar](#calendar)

### **11th 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 P*m*1, P*m*2 and P*m*3, and PCR of P*m* RM1 and P*m* (In)

Electrophoresis of the overlap extension PCR

[Back to the calendar](#calendar)

### **12th February**

**Biofilm Module**

Electrophoresis of P*m* RM1 and P*m* FM1 to compare the concentrations. Dilute 10 times and repeat the overlap extension PCR

Electrophoresis of the overlap extension PCR of P*m*1. It does not go well

[Back to the calendar](#calendar)

### **15th February**

**Biofilm Module**

Inocula of *nahR-*P*sal-nasF-lapG* and *nahR-*P*sal-nasF-yhjH* (complex devices), pUC18Sfi;miniTn7BBGm and *pleD\** in LB

Repeat overlap extension PCR of P*m*1

[Back to the calendar](#calendar)

### **16th 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 P*m*1

[Back to the calendar](#calendar)

### **17th 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 P*m*, P*m*1, P*m*2, P*m*3, *nahR-*P*sal-nasF-lapG* and *nahR-*P*sal-nasF-yhjH*

Ligation of the vector with P*m*, P*m*1, P*m*2 and P*m*3 (P*m* vectors), and Tn7 with *nahR-*P*sal-nasF-lapG* and *nahR-*P*sal-nasF-yhjH* (Tn7 devices)

[Back to the calendar](#calendar)

### **18th February**

**Biofilm Module**

Inocula of each transformation in LB+Km

Transformation with the ligations

[Back to the calendar](#calendar)

### **19th February**

**Biofilm Module**

Miniprep of pleD\* and complex devices

Storage pMRB145, pMRB129 and pMRB130 at -80ºC

[Back to the calendar](#calendar)

### **22nd February**

**Biofilm Module**

Incocula of each transformation in LB

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

Digest pMRB129 and pMRB130 as insert

[Back to the calendar](#calendar)

### **23rd February**

**Biofilm Module**

Miniprep of the cultures

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

Inocula of 124 and pTNS2 in 3 mL of LB

[Back to the calendar](#calendar)

### **24th February**

**Biofilm Module**

Diagnostic digestion of P*m* vectors and Tn7 devices

Electrophoresis of the diagnostic digestions of P*m* vectors and Tn7 devices

Miniprep of the cultures of 124 and pTNS2

Send to sequence the constructions of P*m*

[Back to the calendar](#calendar)

### **25th February**

**Biofilm Module**

Repeat the transformation with Tn7 devices. Now, we name the constructions as pMRB133 (Tn7-*nahR-*P*sal-nasF-lapG*) and pMRB134 (Tn7-*nahR-*P*sal-nasF-yhjH*)

Digest 124, 120 and pSB1K3 as vector

Ligation of 124 (v) with 120 (i) and 127 (i)

Inocula of pMRB125 and pMRB126 in 3 mL of LB

[Back to the calendar](#calendar)

### **26th February**

**Biofilm Module**

Segregate the plates of 133 and 134

Miniprep of the cultures of 125 and 126

Heat-shock transformation with the ligations

Digest 125 and 126 as insert

**Glycerol Module**

PCR *glpF* (primers 1-2, 3-4 and 1-4). Tª annealing 53ºC

Electrophoresis PCR *glpF*. Fragment amplified with primers 3-4 is OK

[Back to the calendar](#calendar)

### **29th 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 P*m*. Now, we name the constructions as pMRB137 (P*m*), pMRB138 (P*m*1), pMRB139 (P*m*2) and pMRB140 (P*m*3)

Electrophoresis of the digestions of 125 and 126, cut a slice of gel that contains the fragment of interest and purify

[Back to the calendar](#calendar)

## **MARCH**

### **1st March**

**The kinetics population model was eventually developed.**

**Biofilm Module**

Miniprep of the cultures

Storage pMRB133 and pMRB134 at -80ºC

Inocula of 124+127 in 3 mL of LB, and each construction of Pm in 5 mL of LB

**Glycerol Module**

Concentration measurement of *glpF* amplified with primers 1-4, using Nanodrop

PCR *glpF* Repetition (primers 1-2, using genomic DNA and fragment amplificated with primers 1-4 as templates, and primers 3-4). Tª annealing 56ºC.

**Propionate Module**

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

[Back to the calendar](#calendar)

### **2nd March**

**Biofilm Module**

Miniprep of the culture

Segregate the plates of the transformations

Digest and electrophoresis of 124+127

Ligation Tn7 with 125 (i) and 126 (i)

**Glycerol Module**

Electrophoresis PCR *glpF*

**Propionate Module**

Transformation of the pSB1K3 plamid into *E. coli* DH5ɑ.

Purification of the *E.coli* K12 genome in order to use it as a template for the PCRs.

[Back to the calendar](#calendar)

### **3rd March**

**Biofilm Module**

Storage pMRB137, pMRB138, pMRB139 and pMRB140 at -80ºC

Transformation of the ligations and pleD\*. Now, we name the construction of pleD\* as pMRB145

Digest 124 as vector

Electrophoresis of the digestion, cut a slice of gel that contains the fragment of interest and purify

Ligation 124 (v) with 120 (i) and 127 (i), and Tn7 with 125 (i) and 126 (i)

**Glycerol Module**

PCR *glpF* with primers 1-2, using genomic DNA as template. Tª annealing 58ºC

**Propionate Module**

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

[Back to the calendar](#calendar)

### **4th 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-gfp*mut3)

**Glycerol Module**

Electrophoresis PCR *glpF* with primers 1-2

**Propionate Module**

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

[Back to the calendar](#calendar)

### **7th March**

**Biofilm Module**

Inocula of 145 in 5 mL of LB

Inocula of pMRB1,124+120 and 124+127 in 3 mL of LB

**Glycerol Module**

PCR *glpF* with primers 1-2. Tª annealing 59ºC

Electrophoresis PCR *glpF*

**Propionate Module**

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

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

[Back to the calendar](#calendar)

### **8th March**

**Biofilm Module**

Storage pMRB145 at -80ºC

Miniprep of the cultures

Digest 145 as vector and 137, 138, 139 and 140 as insert

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

Diagnostic digestion of the pMRB124 constructions

**Glycerol Module**

PCR *glpF* with primers 1-2. Tª annealing 61ºC

**Propionate Module**

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

[Back to the calendar](#calendar)

### **9th March**

**Biofilm Module**

Electrophoresis of the diagnostic digestions

Digest pMRB1 as vector

Repeat the ligation of Tn7 with 125 (i) and 126 (i), and 145 (v) with 120 (i) and 127 (i)

Segregate the plate of 124+127. Now, we name this construction as pMRB146

**Glycerol Module**

Electrophoresis *glpF*

**Propionate Module**

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

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

[Back to the calendar](#calendar)

### **10th 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 P*m* (i)

**Glycerol Module**

PCR *glpF*. Tª annealing 64ºC

**Propionate Module**

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

[Back to the calendar](#calendar)

### **11th March**

**Biofilm Module**

Transformation with the ligations

Plate the strain with 128

Digest of pMRB125 and pMRB126 as insert

**Glycerol Module**

Electrophoresis PCR *glpF*

PCR *glpF* 1-2:

1. 1 μl genomic DNA + 1 μl DMSO. 67ºC

2. 1 μl genomic DNA diluted 1/10. 67ºC

**Propionate Module**

The PCRs of *scABC* were performed to find the optimal conditions.

The *scpC* fragment was obtained (Annealing temp. 54ºC).
Digestion of the *scpA* and *scpC* fragments with XbaI and PstI (overnight).

[Back to the calendar](#calendar)

### **14th March**

**Biofilm Module**

Inocula of 145+120, 145+127, P*m* contructions, 128 and 146

Diagnostic digestion of 145+120 and 145+127

Ligation of Tn7 with 120 (i) and 127 (i), and 124 (v) with 120 (i)

**Glycerol Module**

Electrophoresis PCRs *glpF*

Mix of PCRs *glpF* (45 μl DMSO 67ºC + 45 μl genomic diluted 1/10 67ºC + 45 μl 64ºC

**Propionate Module**

Electrophoresis and purification of the desired bands from both digestions of *scpA* and *scpC*.

The PCR for the *scpB* gene was performed to find the optimal conditions.

[Back to the calendar](#calendar)

### **15th March**

**Biofilm Module**

Miniprep of the cultures and 128 digest as vector

Diagnostic digestion of the P*m* constructions

Storage pMRB146 at -80ºC

Transformation with the ligations

**Glycerol Module**

Geneclean of the band of the fragment of *glpF* amplified with primers 1-2, using the mix created the day 1/7

**Propionate Module**

Ligation of both *scpA* and *scpC* fragments with the digested pSB1K3 plasmid (overnight). The proportions (Plasmid 1:3 Gene) were calculated using the Nanodrop.

The PCR for the *scpB* gene was performed to find the optimal conditions.

[Back to the calendar](#calendar)

### **16th March**

**Biofilm Module**

Electrophoresis of the diagnostic digestions

Electrophoresis of the digestions of 125 (i) and 126 (i), cut a slice of gel that contains the fragment of interest and purify

Ligation of Tn7 with 120, 125 and 126, and 145 (v) with 120 (i)

Inocula of Tn7+127 and 124+120

**Glycerol Module**

Electrophoresis of the geneclean of *glpF* 1-2 and concentration measurement using Nanodrop

**Propionate Module**

The ligation mixtures were transformed into *E. coli* DH5ɑ.

The *scpB* fragment was obtained (Annealing temp. 49 ºC). It was digested overnight using XbaI and PstI.

[Back to the calendar](#calendar)

### **17th March**

**Biofilm Module**

Miniprep of the cultures

Diagnostic digestion of 124+120 and Tn7+127

Transformation with the ligations

**Glycerol Module**

Overlap extension PCR *glpF* (5μl fragment 1-2 + 2,22 μl fragment 3-4 diluted 1/100). 67ºC

Electrophoresis overlap PCR *glpF*

**Propionate Module**

Electrophoresis and purification of the digested *scpB* fragment.

There are no colonies growing after the transformation of the ligation reaction mixture, the cloning process was unsuccessful.

[Back to the calendar](#calendar)

### **18th March**

**Biofilm Module**

The transformations with Tn7 devices didn't work, so plate Tn7 strain again

Transformation with 145+127. Now, we name this construction as pMRB147

**Glycerol Module**

Geneclean overlap PCR *glpF*

Electrophoresis geneclean overlap PCR *glpF*

[Back to the calendar](#calendar)

### **21st March**

**Biofilm Module**

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

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

[Back to the calendar](#calendar)

### **22nd March**

**Biofilm Module**

Miniprep of the cultures

Diagnostic digestion of 145+127 and 145+120

Electrophoresis of pMRB128 (v), cut a slice of gel that contains the fragment of interest and purify

Transformation with pMRB1+137 and Tn7+127

Ligation of Tn7 (v) with 125 (i)

[Back to the calendar](#calendar)

### **23rd March**

**Biofilm Module**

Inocula of pMRB1+137

Transformation with Tn7+pMRB125

Electrophoresis of the diagnostic digestion of 145+120

[Back to the calendar](#calendar)

### **24th March**

**Biofilm Module**

Minipreps and diagnostic digestion of 1+137

Segregate the plate of Tn7+127

Digestion of 145 and 147 as an insert

Ligation of Tn7 (v) with 125 (i) and 126 (i), and 145 (v) with 120 (i)

[Back to the calendar](#calendar)

### **25th March**

**Biofilm Module**

Transformation of the ligations

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

Diagnostic digestion of pMRB1+137/138/139/140

[Back to the calendar](#calendar)

### **28th March**

**Biofilm Module**

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

The transformations did not go well

[Back to the calendar](#calendar)

### **29th March**

**Biofilm Module**

Ligation of 120 (v) with 124 (i), Tn7 (v) with 125 (i), 147 (i) and 126 (i), pMRB1 (v) with 137-140 (i) and 145 (v) with 120 (i)

Store at -80ºC and minipreps of pMRB151

[Back to the calendar](#calendar)

### **30th March**

**Biofilm Module**

Transformation of the ligations

[Back to the calendar](#calendar)

### **31th March**

**Biofilm Module**

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

Segregate the transformation of Tn7+126

[Back to the calendar](#calendar)

## **APRIL**

### **1st April**

**Biofilm Module**

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

Electrophoresis of the diagnostic digestion

[Back to the calendar](#calendar)

### **4nd April**

**The kinetics population model was tested using carbon source as limiting substrate.**

**Biofilm Module**

2nd diagnostic digestions of 1+137/138/139/140

Inocula of 124,135 and 136

Transformation of 145+120 and Tn7+147. Now, we name these constructions as pMRB135 and pMRB136, respectively

**Glycerol Module**

Overlap PCR *glpF* 71ºC and electrophoresis

**Propionate Module**

The ligation reaction was performed overnight again for *scpAC* and also for *scpB*, using new proportions this time. The proportions (Plasmid 1:6 Gene) were calculated using the Nanodrop.

[Back to the calendar](#calendar)

### **5th April**

**Biofilm Module**

Storage pMRB135 and pMRB136 at -80ºC

Inocula of KT2442

Digest 135,145 and 130 as insert and Tn7 as vector

Measure the concentration of pTNS2, pMRB136 and pMRB134>

**Glycerol Module**

Precipitation of overlap PCR of *glpF* and concentration measurement using Nanodrop

**Propionate Module**

Transformation of the ligation into *E. coli* DH5ɑ.

[Back to the calendar](#calendar)

### **6th April**

**Biofilm Module**

Electrophoresis of digestion of 135, 145, 130 and Tn7, cut a slice of gel that contains the fragment of interest and purify

Plate the strains with pMRB136 and pTNS2

Electroporation of KT2442 with 136 and 134

**Glycerol Module**

Overlap PCR of *glpF* 71ºC (again) and electrophoresis. There is nothing!

**Propionate Module**

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

[Back to the calendar](#calendar)

### **7th April**

**Biofilm Module**

Inocula of pTNS2 and 136

Ligation of Tn7 with 126 and 135

Colony PCR of the electroporations

**Glycerol Module**

PCR *glpF* 1-2:

1. 1 μl genomic DNA + 1 μl DMSO. 67ºC

2. 1 μl genomic DNA diluted 1/10. 67ºC

Electrophoresis PCRs *glpF* 1-2

**Propionate Module**

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

[Back to the calendar](#calendar)

### **8th April**

**Biofilm Module**

Minipreps of pTNS2 and 136

Transformation of the ligations

Electrophoresis of the colony PCR

**Glycerol Module**

Geneclean of the PCRs 1-2 and nanodrop measurement

**Propionate Module**

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

[Back to the calendar](#calendar)

### **11th April**

**Biofilm Module**

Inocula of Tn7+126 and Tn7+135

Ligation of 1 (v) with 138 (i), 124 (v) with 120 (i) and Tn7 (v) with 120 (i) and 125 (i)

**Glycerol Module**

Electrophoresis geneclean PCR *glpF* 1-2

Overlap PCR *glpF* (using fragment 1-2 obtained the day 24/1)

Electrophoresis of overlap PCR *glpF*

**Propionate Module**

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

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

[Back to the calendar](#calendar)

### **12th April**

**Biofilm Module**

Minipreps and diagnostic digestion of the cultures

Transformation of the ligations

**Glycerol Module**

*glpF* fragment (overlap PCR precipitated the day 31/1) digestion using XbaI and PstI restriction enzymes

**Propionate Module**

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

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

[Back to the calendar](#calendar)

### **13th April**

**Biofilm Module**

Inocula of 1+138, 124+120, and Tn7+125. Now ,we name 124+120 as pMRB152

Ligation of Tn7 (v) with 130 (i), 145 (i), 146 (i) and 120 (i)

**Glycerol Module**

Heat inactivation restriction enzymes (20’ 80ºC)

Measurement of the concentration of *glpF* digested using nanodrop

**Propionate Module**

Electrophoresis and purification of the desired band of pSB1K3.

Ligation reaction with the three *scp* genes, this time with the 1:6 proportions (overnight).

[Back to the calendar](#calendar)

### **14th April**

**Biofilm Module**

Minipreps of the cultures

Storage pMRB152 at -80ºC

Diagnostic digestion and electrophoresis of 1+137/138/139/140, Tn7+125, Tn7+135 and Tn7+126

Transformation with Tn7+120, Tn7+145, Tn7+130, Tn7+146 and Tn7+125

**Glycerol Module**

Digested pSB1K3 obtaining and electrophoresis

**Propionate Module**

Transformation of the ligation mixture into *E. coli* DH5ɑ.

[Back to the calendar](#calendar)

### **15th April**

**Biofilm Module**

Measure the concentration of 136 and 134

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

Plate the strain with pTNS2

**Glycerol Module**

Geneclean of the fragment of the plasmid that will be used to clone *glpF* and measurement of the concentration using nanodrop

**Propionate Module**

Two colonies are obtained for *scpA* and one for *scpC*.

[Back to the calendar](#calendar)

### **18th April**

**Biofilm Module**

Inocula of KT2442, pTNS2, Tn7+145, Tn7+130, Tn7+146, Tn7+120 and Tn7+125. Now, we name Tn7+125 as pMRB128, Tn7+130 as pMRB164 and Tn7+145 as pMRB165

Electrophoresis of the diagnostic digestions

**Glycerol Module**

Ligation of *glpF* to pSB1K3

**Propionate Module**

Liquid cultures are established from the colonies.

[Back to the calendar](#calendar)

### **19th April**

**Biofilm Module**

Electroporation of KT2442 with 164, 165, 134 and 136

Minipreps of pTNS2, Tn7+146 and Tn7+120

Storage pMRB128, pMRB164 and pMRB165 at -80ºC

Transformation of 1+Pms. Now, we name 1+Pm, 1+Pm1, 1+Pm2 and 1+Pm3 as pMRB166, pMRB167, pMRB168 and pMRB169

**Glycerol Module**

Transformation of the ligation to bacteria (E. coli DH5α)

**Propionate Module**

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

[Back to the calendar](#calendar)

### **20th April**

**Biofilm Module**

Colony PCR of the electroporations and electrophoresis

Diagnostic digestion of Tn7+146 and Tn7+120

Segregate the positive candidates of electroporations

Plate KT2442-Tn7

Inocula of 166, 167, 168 and 169

**Glycerol Module**

White colonies obtained. 3 inocula of the colonies of pSB1K3-*glpF*

**Propionate Module**

New primers are designed and ordered in order to amplify the whole propionate operon. A mutagenic PCR is required (two pairs of primers needed).

[Back to the calendar](#calendar)

### **21st April**

**Biofilm Module**

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

Electrophoresis of Tn7+146 and Tn7+120

Diagnostic digestion of 120 and 146

Storage at -80ºC and minipreps of pMRB166-169

**Glycerol Module**

Minipreps of the plasmids

Digestion of the plasmids

[Back to the calendar](#calendar)

### **22nd April**

**Biofilm Module**

Digest 152 as insert

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

**Glycerol Module**

Electrophoresis of the digested samples. No good results. There was no a band with the size of *glpF*

[Back to the calendar](#calendar)

### **25th April**

**Biofilm Module**

Plate the strains KT2442-Tn7, KT2442-T134, KT2442-T136, KT2442-T164 and KT2442-T165

Inocula for fluorimetry

Electrophoresis of 152 (i), cut a slice of gel that contains the fragment of interest and purify

**Glycerol Module**

PCR of the rest of colonies of the plaque of the ligation of *glpF* to pSB1K3, using Taq polymerase

**Propionate Module**

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

[Back to the calendar](#calendar)

### **26th April**

**Biofilm Module**

Digest pSB1K3 as vector

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

Fluorimeter

**Glycerol Module**

Electrophoresis of the PCR of *glpF*. There are positive clones (numbers 6, 8, 11, 12 and 14)

**Propionate Module**

Electrophoresis in order to check the PCR results. The temperature must be increased. The PCRs are performed again. Positive results for fragment A (69ºC).

[Back to the calendar](#calendar)

### **27th April**

**Biofilm Module**

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

Failed fluorimeter

**Glycerol Module**

Inocula of the positive clones of pSB1K3-*glpF*. 2 inocula of 8 and 12 (they will be sequenced)

**Propionate Module**

The PCR of the fragment B is performed again.

Electrophoresis of fragment A, purification of the desired band.

[Back to the calendar](#calendar)

### **28th 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-*glpF*8, pSB1K3-*glpF*12 and pSB1K3-*glpF*14)

Minipreps of pSB1K3-*glpF8* and pSB1K3-*glpF12*

**Propionate Module**

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

[Back to the calendar](#calendar)

### **29th April**

**Biofilm Module**

Transformation of the ligations

Plate 128, 133, KT2442-Tn7/T136/T165 and KT2442, KT2442 *lapG*- and KT2442 Δ*bifA*

**Propionate Module**

The *epi* gene is ordered to a gene synthesis company.

[Back to the calendar](#calendar)

## **MAY**

### **2nd May**

**The kinetics population model was tested using oxygen as limiting substrate.**

**Biofilm Module**

Inocula of Tn7+146, Tn7+152, KT2442 *lapG*-, 128 and 133

Plate all the KT2442 with pCdrA, KT2442 ΔfleQ and all the variants of KT2442 of pleD\*

Electrophoresis of 120 (i), cut a slice of gel that contains the fragment of interest and purify

[Back to the calendar](#calendar)

### **3rd May**

**Biofilm Module**

Minipreps and measure the concentration of 128 and 133

Minipreps and diagnostic digestion of Tn7+146 and Tn7+152

Electroporation of KT2442 *lapG*- with 128 and 133

**Glycerol Module**

Concentration measurement of plasmids 8 and 12 using nanodrop. Send to secugen to sequence

**Propionate Module**

Measured concentration of the digested A and B fragments using NanoDrop. Both are combined for the final PCR. Optimal conditions must be obtained.

[Back to the calendar](#calendar)

### **4th May**

**Biofilm Module**

Inocula for curves and fluorimeter

Electrophoresis of the diagnostic digestions

Dilutions of fluorimeter and curves of KTs

**Glycerol Module**

Digestion of the plasmids 8 and 12 with XbaI and PstI restriction enzymes

**Propionate Module**

PCR unsuccessful. The temperature is decreased.

The result is negative again and a temperature gradient is used.

Transformation of more pSB1K3 plasmid into *E. coli* DH5ɑ.

[Back to the calendar](#calendar)

### **5th May**

**Biofilm Module**

Dye and measure the curves of pleD\*

Take the data of the fluorimeter

**Glycerol Module**

Electrophoresis of the digestions

Spread bacteria (frozen at -80ºC) with plasmids 8 and 12 on agar plates, and let them grow at 37ºC

**Propionate Module**

A mistake is detected in the primers that were been used (the overlapping sequence is too short). New primers are designed and ordered.

Liquid cultures of the colonies obtained from the transformation of pSB1K3.

[Back to the calendar](#calendar)

### **6th May**

**Biofilm Module**

Plate the variants of KT2442 with yhjH

Transformation of Tn7+120

**Glycerol Module**

Spread the bacteria grown at 37ºC on other plaques and let them grow at room temperature

**Propionate Module**

Miniprep of the pSB1K3 plasmid.

[Back to the calendar](#calendar)

### **9th May**

**Biofilm Module**

Inocula of KT2442-Tn7/T134/T164 and Tn7+120

Transformation with Tn7+146 and Tn7+152. Now, we name these strains as pMRB159 and pMRB170, respectively

**Glycerol Module**

Inocula using the plaques of the day 3/3. One of each plasmid (8 and 12)

**Propionate Module**

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

[Back to the calendar](#calendar)

### **10th May**

**Microscopic simulation of biofilm growth model was eventually developed.**

**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-*glpF*8 and pSB1K3-*glpF12*

**Propionate Module**

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

[Back to the calendar](#calendar)

### **11th 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-*glpF*8, pSB1K3-*glpF*12 and pSB1K3 with XbaI and PstI restriction enzymes

**Propionate Module**

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

[Back to the calendar](#calendar)

### **12th 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-*glpF*8 is OK!

![](https://static.igem.org/mediawiki/2016/1/12/T--UPO-Sevilla--gel125.jpg)

**Propionate Module**

A PCR is performed with equal amounts of each fragment. The result is negative. A temperature gradient is performed but the results are again negative.

[Back to the calendar](#calendar)

### **13th May**

**Biofilm Module**

PCR of pMRB1 to get the gene *gfp*

Electrophoresis of the colony PCRs

Segregate Tn7+120

**Propionate Module**

A PCR without primers is performed at 72ºC using high amounts of both fragments. A bit of DNA from the operon is obtained.

[Back to the calendar](#calendar)

### **16th May**

**Biofilm Module**

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

Inocula of Tn7+120

Colony PCR and electrophoresis of KT2442 *lapG*--T133

**Propionate Module**

The band corresponding to the whole mutated operon is purified using silica beads, but the yield is too low and the DNA is lost.

The PCR is performed again.

[Back to the calendar](#calendar)

### **17th May**

**Biofilm Module**

Storage KT2442 *lapG*--T128 at -80ºC

Dilutions of pleD\* curves

Plate KT2442 *lapG*- and KT2442 Δ*bifA*

Inocula of KT2442

Inocula of KT2442 and KT2442-Tn7/T134/T136/T164/T165 for Congo Red and curves

**Glycerol Module**

Send pSB1K3-*glpF*8 to Secugen to be sequenced

**Propionate Module**

Negative results for the PCR although same conditions have been used.

A concentration gradient for each of the fragments is used for a new PCR.

[Back to the calendar](#calendar)

### **18th 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 Δ*bifA*

Plates of Congo Red

Curves of 134, 164 and KT2442

**Glycerol Module**

pSB1K3-*glpF*8 digestion using XbaI and PstI restriction enzymes

**Propionate Module**

Negative result for the PCR. New conditions for temperature (69ºC) and concentration of the fragments A and B are tested (without primers), and some amount of DNA corresponding to the operon is obtained.

[Back to the calendar](#calendar)

### **19th May**

**Biofilm Module**

Electrophoresis of the PCR of *gfp*

Curves of 165

Electroporation of KT2442 *lapG*- and Δ*bifA* with Tn7, 128 and 133

Dye and measure the plates of 134, 164 and KT2442

Let more time the plates with Congo Red

**Glycerol Module**

Electrophoresis of the digested sample and purification of the band of *glpF*

**Propionate Module**

More PCR tubes are used in order to get more DNA. Electrophoresis and purification of the band corresponding to the operon.

This DNA is digested using XbaI and PstI overnight.

[Back to the calendar](#calendar)

### **20th May**

**Biofilm Module**

Dye and measure the plates of 165 at 17 h and 20 h

Take a photo of the plates with Congo Red

Let the plates with Congo Red at RT overnight

**Glycerol Module**

Electrophoresis of the geneclean. OK!

**Propionate Module**

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

[Back to the calendar](#calendar)

### **23rd 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-*glpF*8 digestion and geneclean

Take out frozen bacteria with pSB1K3-*glpF*8 and let them grow in a liquid medium at 37ºC

**Propionate Module**

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

[Back to the calendar](#calendar)

### **24th 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 Δ*bifA*-T128

**Glycerol Module**

Put the flask at 4ºC of the bacteria growing in a liquid medium

**Propionate Module**

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

[Back to the calendar](#calendar)

### **25th May**

**Biofilm Module**

Inocula of the plates of yesterday

Colony PCR and electrophoresis of KT2442 *lapG*--Tn7/T133, Δ*bifA*-T128/T133 and KT2442-T133

Storage KT2442-T170/T159/T128, *lapG*--Tn7 and Δ*bifA*-T128 at -80ºC

**Glycerol Module**

Results of the sequentiation of *glpF*. IT IS OK!!!

Centrifugation of the flask and freeze the pellet at -80ºC

**Propionate Module**

Miniprep of the pUC57 plasmid. Digestion with EcoRI and SpeI overnight.

A stock of the pSB1K3 plasmid is digested with EcoRI and SpeI overnight.

[Back to the calendar](#calendar)

### **26th 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 Δ*bifA*-T133

**Glycerol Module**

Electrophoresis of the purified band of pSB1K3-*glpF8*

**Propionate Module**

Electrophoresis and purification of the desired band (epi gene from pUC57 and the backbone from pSB1K3).

Ligation of both fragments, overnight using the proportions vector 1:3 gene.

Ligation of the operon fragment with the digested pSB1K3 backbone.

[Back to the calendar](#calendar)

### **27th 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-*glpF*8 (frozen pellet at -80ºC)

**Propionate Module**

Transformation of the ligation mix for the *epi* gene and the operon. The plates are left at room temperature for the whole weekend.

[Back to the calendar](#calendar)

### **30th 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*/P*sal*/*nasF*) digestion with SpeI and PstI and electrophoresis

**Propionate Module**

Seven colonies are obtained from the transformation of the ligation of *epi*. Liquid cultures are established from all of them.

The ligation of the operon gives negative results.

[Back to the calendar](#calendar)

### **31th May**

**Biofilm Module**

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

Plate pMRB133

**Glycerol Module**

Geneclean of pMRB151 digested. Purify the fragment that will be used for the ligation

**Propionate Module**

Miniprep from the liquid cultures. Digestion with EcoRI and PstI: all the clones are positive for *epi*. However, due to restriction sites issues there is a fragment of the pUC57 vector cloned next to the gene. A new ligation must be done with the DNA from the pSB1K3-epi vector.

[Back to the calendar](#calendar)

## **JUNE**

### **1st June**

**Biofilm Module**

Dilutions and prepare curves of KT2442-T134/T164 and KT2442-Tn7/T128 and *lapG*--Tn7/T128

Inocula of KT2442-Tn7/T128 and *lapG*--Tn7/T128

**Glycerol Module**

Electrophoresis of the geneclean and measurement of the concentration using nanodrop

Ligation of pMRB151 and *glpF* (1:3 proportion)

**Propionate Module**

The clone number 1 of the pSB1K3-*epi* plasmid was digested overnight with XbaI and PstI in order to obtain the gene fragment.

[Back to the calendar](#calendar)

### **2nd June**

**Biofilm Module**

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

Dilutions and prepare curves of KT2442-Tn7/T128 and *lapG*--Tn7/T128

Inocula of 133

**Glycerol Module**

Transformation of half the volume of the ligation

**Propionate Module**

Electrophoresis and purification of the band corresponding to the *epi* gene. A new ligation reaction is set with the digested pSB1K3 backbone (overnight).

[Back to the calendar](#calendar)

### **3rd June**

**Biofilm Module**

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

Miniprep and measure the concentration of 133

**Glycerol Module**

There is nothing!

**Propionate Module**

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

[Back to the calendar](#calendar)

### **6th June**

**Biofilm Module**

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

Diagnostic digestion and electrophoresis of 133

**Glycerol Module**

Transformation of the rest of the ligation sample

**Propionate Module**

Six colonies are obtained, and liquid cultured are prepared for all colonies.

A new PCR of the operon is performed in order to obtain more DNA (fragments A and B).

[Back to the calendar](#calendar)

### **7th June**

**Biofilm Module**

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

Electroporation of KT2442, KT2442 *lapG*- and Δ*bifA* with pMRB133

**Glycerol Module**

There is nothing!

Digestion of the geneclean of pMRB151

**Propionate Module**

Miniprep from the liquid cultures. Digestion with XbaI and PstI: all the clones are positive for epi. The construct is finally ready.

Electrophoresis of fragments A and B of the operon and purification of the desired bands.

[Back to the calendar](#calendar)

### **8th 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*-/Δ*bifA*-T133

**Glycerol Module**

Electrophoresis of the digestion

**Propionate Module**

The new pSB1K3 (clone number 1) vector is digested with XbaI and PstI (overnight).

The plasmids with the expression system (nahR-Psal) and miniTn7, pMRB172 and pMRB151, are digested overnight with PstI and SpeI.

PCR of the operon using the same conditions as in the last time (same temperature and concentration of fragments A and B).

[Back to the calendar](#calendar)

### **9th June**

**Biofilm Module**

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

**Glycerol Module**

Geneclean of digested pMRB151. Purify the fragment that will be used for the ligation

**Propionate Module**

Electrophoresis and purification of the desired bands (the *epi* gene and the two vector backbones from pMRB172 and pMRB151. The band corresponding to the operon from the PCR is also purified.

Two ligations reactions are set overnight (with controls) for the *epi* gene and each vector backbone.

[Back to the calendar](#calendar)

### **10th June**

**Biofilm Module**

Storage KT2442/*lapG*-/Δ*bifA*-T133 at -80ºC

**Glycerol Module**

Electrophoresis of the purified band. There is nothing!

[Back to the calendar](#calendar)

### **13th June**

**Biofilm Module**

Plate *Pseudomonas putida* strains hosting pMRB134, 136, 164 & 165, as well as the wild type hosting the miniTn7 device empty.

**Glycerol Module**

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

**Propionate Module**

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

[Back to the calendar](#calendar)

### **14th June**

**Biofilm Module**

Inocula of all the strains plated yesterday. Two different colonies were picked in order to have two different biological repeats.

Make Congo Red plates by adding Congo Red at a concentration of 40 mg/ml in T-medium agar.

**Glycerol Module**

3 inocula of pMRB151 (A, B and C)

**Propionate Module**

Many colonies are obtained for each ligation. Six colonies are obtained for pMRB172 and one for pMRB151.

Liquid cultures are set for each colony.

[Back to the calendar](#calendar)

### **15th June**

**Biofilm Module**

Inoculate 10 micro liters of culture media from the inocula. Let it dry in the hood and incubate at 30 degrees overnight.

**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-*glpF*8

Electrophoresis of all digestions

Repeat diagnostic digestions

**Propionate Module**

Miniprep from the liquid cultures. Digestion of the plasmids using XbaI and PstI. The resulting bands correspond to the size of the *epi* gene + the expression module. Therefore they are positive.

[Back to the calendar](#calendar)

### **16th June**

**Biofilm Module**

See the plates of Congo Red. They need almost 24 hours more of growing.

**Glycerol Module**

Electrophoresis of all digestions (again)

**Propionate Module**

[Back to the calendar](#calendar)

### **17th June**

**Biofilm Module**

See the Congo Red plates and take photographs of them.

**Glycerol Module**

Geneclean pSB1K3-*glpF*8 digested

pSB1K3-*glpF*8 digestion with EcoRI and XbaI

Spread bacteria with pSB1K3-*glpF*8 and incubate at room temperature

[Back to the calendar](#calendar)

### **20th June**

**Biofilm Module**

We are going to repeat the Congo Red experiments.

Plate *Pseudomonas putida* strains hosting pMRB134, 136, 164 & 165, as well as the wild type hosting the miniTn7 device empty.

**Glycerol Module**

Electrophoresis

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

**Propionate Module**

The clone number 1 of the pSB1K3-epi plasmid was digested overnight with EcoRI and PstI in order to obtain the gene fragment.

Transformation of the pSB1C3 plasmid into *E. coli* DH5ɑ.

[Back to the calendar](#calendar)

### **21st June**

**Biofilm Module**

Inocula of all the strains plated yesterday. Two different colonies were picked in order to have two different biological repeats.

Make Congo Red plates by adding Congo Red at a concentration of 40 mg/ml in T-medium agar.

**Glycerol Module**

Minipreps of pSB1K3-*glpF*8 A, B, C, D and E and digestions with EcoRI and PstI restriction enzymes

Electrophoresis

DNA precipitation and electrophoresis

**Propionate Module**

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

Liquid cultures from the colonies from the transformation of pSB1C3.

[Back to the calendar](#calendar)

### **22nd June**

**Biofilm Module**

Inoculate 10 micro liters of culture media from the inocula. Let it dry in the hood and incubate at 30 degrees overnight.

**Glycerol Module**

pSB1K3-*glpF*8 A, B, C, D and E digestions

Inocula of pMRB151 A, B and C

Geneclean of pSB1K3-*glpF*8 A-E. Purify the band that corresponds to *glpF*

**Propionate Module**

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

[Back to the calendar](#calendar)

### **23rd June**

**Biofilm Module**

See the plates of Congo Red. They need almost 24 hours more of growing.

**Glycerol Module**

Minipreps of pMRB151 A, B and C

glpF8 geneclean

pMRB172 (mini-Tn7-*nahR*-P*sal*) obtaining

**Propionate Module**

Electrophoresis and purification of the desired bands (the backbone from pSB1C3).

A new ligation reaction is set with the digested pSB1C3 backbone and the *epi* gene (overnight).

[Back to the calendar](#calendar)

### **24th June**

**Biofilm Module**

See the Congo Red plates and take photographs of them.

**Glycerol Module**

pMRB151 and pMRB172 digestions

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

Repeat digestion of pMRB151

**Propionate Module**

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

[Back to the calendar](#calendar)

### **27th June**

**Biofilm Module**

Refresh strains from last week.

Make swimming plates.

**Glycerol Module**

Electrophoresis of the genecleans of pMRB151 and ppMRB172

Ligations of *glpF* to pMRB172 and pMRB151

**Propionate Module**

There are no colonies growing in the plates. These are incubated at 37ºC overnight.

The blunt pJET vector is used in order to set a ligation reaction for the operon fragment (the protocol by CloneJET is used). The ligation mixture is transformed into *E. coli* DH5ɑ.

[Back to the calendar](#calendar)

### **28th June**

**Biofilm Module**

Pick two different colonies from each strain and inoculate them in swimming plates both with and without salicylate.

Incubate the plates for 12 hours at 30 degrees..

**Glycerol Module**

Transformations of the ligations to bacteria

**Propionate Module**

Again, negative results for the ligation of *epi* and pSB1C3.

Many colonies are obtained for the pJET ligation. 10 colonies are used for liquid cultures.

[Back to the calendar](#calendar)

### **29th June**

**Biofilm Module**

Look at the halos produced by the bacteria and photograph them.

Inoculate two more colonies from the strains in new swimming plates in order to have a second technical repeat of the experiments.

**Glycerol Module**

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

**Propionate Module**

Miniprep of the liquid cultures.

Digestion in order to check if the operon has been cloned. Negative result.

Another 10 colonies from the ligation in pJET are used for a liquid culture.

[Back to the calendar](#calendar)

### **30th June**

**Biofilm Module**

Look at the halos produced by the swimming bacteria and photograph them.

**Glycerol Module**

Electrophoresis of the PCRs

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

**Propionate Module**

Miniprep of the liquid cultures.

Digestion in order to check if the operon has been cloned. Again, negative result.

The three genes in the operon, *scpABC*, are ordered to IDT using the special offer, due to the impossibility to clone them by ourselves.

[Back to the calendar](#calendar)

## **JULY**

### **1st July**

**Glycerol Module**

Freeze all bacteria (for storage)

Minipreps of the rest of inocula

Electrophoresis of minipreps

Diagnostic digestions of the plasmids

[Back to the calendar](#calendar)

### **4th July**

**Biofilm Module**

Plate the strains hosting the miniTn7 devices pMRB134, 136, 164 % 165 as well as the wild type strain hosting the miniTn7 device empty.

**Glycerol Module**

Electrophoresis of all 4 digestions. PERFECT!

Measurement of the concentration using nanodrop for electroporation

![](https://static.igem.org/mediawiki/2016/7/77/T--UPO-Sevilla--gel47.jpg)

[Back to the calendar](#calendar)

### **5th July**

**Biofilm Module**

Inoculate two colonies of each strain in bactotryptone media at 0,3%.

Incubate the inocula at 30 degrees and 180 rpm overnight.

**Glycerol Module**

Obtaining of *Pseudomonas putida* KT2442 and inoculate it in LB medium

[Back to the calendar](#calendar)

### **6th July**

**Biofilm Module**

Look at the formation of pellicle in the walls of the tube and photograph them.

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **7th July**

**Biofilm Module**

We repeat the experiment. Inoculate two colonies of each strain in bactotryptone media at 0,3%.

**Glycerol Module**

PCR of the colonies obtained in the electroporation and electrophoresis

4 Inocula of the positive clones, 2 of each electroporation

![](https://static.igem.org/mediawiki/2016/3/35/T--UPO-Sevilla--gel77.jpg)

[Back to the calendar](#calendar)

### **8th July**

**Biofilm Module**

Look at the formation of pellicle in the walls of the tube and photograph them.

**Glycerol Module**

Freeze all four bacteria

[Back to the calendar](#calendar)

### **11th July**

**Biofilm Module**

Refresh all the strains from last week hosting the miniTn7 devices.

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **12th July**

**Biofilm Module**

Inocula of two different colonies from yesterday's strains.

**Glycerol Module**

3 inocula of KT2442 with pMRB172-*glpF* and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source

[Back to the calendar](#calendar)

### **13th July**

**Biofilm Module**

Adhesion assay (see Protocols).

**Glycerol Module**

Experiment 1: Preparation of the plaques and fluorometer starting

[Back to the calendar](#calendar)

### **14th July**

**Biofilm Module**

In order to repeat the adhesion experiment we do inocula of the strains hosting the miniTn7 device.

**Glycerol Module**

Measurement of experiment 1

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

[Back to the calendar](#calendar)

### **15th July**

**Biofilm Module**

Adhesion assay (see Protocols).

**Glycerol Module**

Measurement of Experiment 2

[Back to the calendar](#calendar)

### **18th July**

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **19th July**

**Glycerol Module**

3 inocula of KT2442 with pMRB172-*glpF* and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source

[Back to the calendar](#calendar)

### **20th July**

**Glycerol Module**

Experiment 3: Preparation of the plaques and fluorometer starting

[Back to the calendar](#calendar)

### **21st July**

**Glycerol Module**

Measurement of Experiment 3

[Back to the calendar](#calendar)

### **22nd July**

[Back to the calendar](#calendar)

### **25th July**

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **26th July**

**Glycerol Module**

3 inocula of KT2442 with pMRB172-*glpF* and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source

[Back to the calendar](#calendar)

### **27th July**

**Glycerol Module**

Experiment 4: Preparation of the plaques and fluorometer starting

[Back to the calendar](#calendar)

### **28th July**

**Glycerol Module**

Measurement of Experiment 4

[Back to the calendar](#calendar)

### **29th July**

[Back to the calendar](#calendar)

## **AUGUST**

### **1st August**

**Biofilm Module**

Plate KT2442/*lapG*-/Δ*bifA*-Tn7/T133

Plate pMRB1, 166, 167, 168, 169, KT2442-T170/T159 and pRK2013 (plasmid helper for triparental mating)

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **2nd August**

**Biofilm Module**

Inocula of 166, 167, 168, 169, KT2442-T170/T159 and pRK2013

**Glycerol Module**

3 inocula of KT2442 with pMRB172-*glpF* and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source

[Back to the calendar](#calendar)

### **3rd August**

**Biofilm Module**

Inocula of KT2442/*lapG*--Tn7/T133

Triparental mating of KT2442-T170 and KT2442-T159 with pMRB1, 166, 167, 168 and 169

**Glycerol Module**

Experiment 5: Preparation of the plaques and fluorometer starting

[Back to the calendar](#calendar)

### **4th August**

**Biofilm Module**

Dilutions and prepare the curves of KT2442/*lapG*--Tn7/T133 for 20 h

Spread the plates of triparental mating onto plates with Cb (Carbamicilin) and Gm (Gentamicin)

**Glycerol Module**

Measurement of Experiment 5

[Back to the calendar](#calendar)

### **5th August**

**Biofilm Module**

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

[Back to the calendar](#calendar)

### **8th August**

**Biofilm Module**

Inocula of KT2442/*lapG*--Tn7/T133

Segregate the plates of triparental mating

**Glycerol Module**

Digestion of pMRB165 (mini-Tn7-*nahR*-P*sal*-*pleD\**) with SpeI and PstI restriction enzymes

Digestion of pSB1K3-*glpF* with XbaI and PstI restriction enzymes

[Back to the calendar](#calendar)

### **9th August**

**Biofilm Module**

Dilutions and prepare the curves of KT2442/*lapG*--T133 for 20 h

Inocula of KT2442/*lapG*--Tn7/T133

Inocula of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169

**Glycerol Module**

Electrophoresis and geneclean of the digestions of pMRB165 and pSB1K3-*glpF*

Electrophoresis of the purified bands

Repeat digestion of pSB1K3-*glpF*

[Back to the calendar](#calendar)

### **10th August**

**Biofilm Module**

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

Dilutions and prepare the curves of KT2442/*lapG*--T133 for 20 h

Storage KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 at -80ºC

**Glycerol Module**

Electrophoresis and geneclean of pSB1K3 to purify *glpF*

Electrophoresis of the geneclean

[Back to the calendar](#calendar)

### **11th August**

**Biofilm Module**

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

[Back to the calendar](#calendar)

### **12th August**

[Back to the calendar](#calendar)

### **15th August**

**Biofilm Module**

Inocula of Δ*bifA*-Tn7/T128

**Glycerol Module**

Ligation of *glpF* in pMRB165

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

[Back to the calendar](#calendar)

### **16th August**

**Biofilm Module**

Dilutions and prepare the curves of Δ*bifA*-Tn7/T128 for 20 h

**Glycerol Module**

Transformation of the ligation

3 inocula of KT2442 with pMRB172-*glpF* and other 3 of KT2442 with Tn7 Ø in minimal media with succinate as the carbon source

[Back to the calendar](#calendar)

### **17th August**

**Biofilm Module**

Dye and measure the plates of Δ*bifA*-Tn7/T128

**Glycerol Module**

Experiment 6: Preparation of the plaques and fluorometer starting

5 inocula of the colonies obtained after the transformation of the ligation

[Back to the calendar](#calendar)

### **18th August**

**Glycerol Module**

Measurement of Experiment 6

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

[Back to the calendar](#calendar)

### **19th August**

**Biofilm Module**

Plate KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 for beta-galactosidase assay

**Glycerol Module**

Digestions of pMRB165-*glpF* with XbaI and PstI restriction enzymes

Electrophoresis of the digestions. It is OK!

[Back to the calendar](#calendar)

### **22nd August**

**Biofilm Module**

Inocula of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169

**Glycerol Module**

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

[Back to the calendar](#calendar)

### **23rd August**

**Biofilm Module**

Dilutions of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169 for beta-galactosidase assay

**Glycerol Module**

1 inocula of KT2442 with pMRB172-*glpF* and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source

[Back to the calendar](#calendar)

### **24th August**

**Biofilm Module**

Beta-galactosidase assay (the strains with pMRB166 have not worked, it is the original Pm)

Inocula of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169

**Glycerol Module**

Dilutions and preparation of the microtiter plates

[Back to the calendar](#calendar)

### **25th August**

**Biofilm Module**

Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay

**Glycerol Module**

Experiment 7: Measurement of planktonic cells and biofilms

[Back to the calendar](#calendar)

### **26th August**

**Biofilm Module**

Beta-galactosidase assay

Segregate the plates of KT2442-T159-1/166/167/168/169 and KT2442-T170-1/166/167/168/169

[Back to the calendar](#calendar)

### **29th August**

**Biofilm Module**

Inocula of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169

**Glycerol Module**

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

**Migration to pSB1C3**

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

[Back to the calendar](#calendar)

### **30th August**

**Biofilm Module**

Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay

**Glycerol Module**

1 inocula of KT2442 with pMRB172-*glpF* and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source

**Migration to pSB1C3**

Inocula of yesterday plates to do minipreps.

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

[Back to the calendar](#calendar)

### **31th 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).

![](https://static.igem.org/mediawiki/2016/thumb/c/c9/T--UPO-Sevilla--Primero.jpeg/800px-T--UPO-Sevilla--Primero.jpeg.png)

Ligation of all the fragments with pSB1C3.

Inocula of yesterday’s plates (123, 137, and *glpF*).

[Back to the calendar](#calendar)

## **SEPTEMBER**

### **1st September**

**Biofilm Module**

Dilutions of KT2442-T159-1/167/168/169 and KT2442-T170-1/167/168/169 for beta-galactosidase assay

**Glycerol Module**

Experiment 8: Measurement of planktonic cells and biofilms

**Migration to pSB1C3**

Transformation of ligation of fragments of 124, 125, 126, 135, 139, 145, 147 & 152.

Digestion of 122 with EcoRI-PstI.

Preparative electrophoresis gel. Isolation of the band from 122 fragment.

![](https://static.igem.org/mediawiki/2016/thumb/d/d0/T--UPO-Sevilla--122.png/448px-T--UPO-Sevilla--122.png.jpeg)

Ligation of 122 with C3.

Kit minipreprs of yesterday’s inocula (123, 137, and *glpF*).

Digestion of the inserts of the plasmids with EcoRI-PstI.

[Back to the calendar](#calendar)

### **2nd 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*.

![](https://static.igem.org/mediawiki/2016/thumb/1/10/T--UPO-Sevilla--123.png/448px-T--UPO-Sevilla--123.png.jpeg)
![](https://static.igem.org/mediawiki/2016/thumb/c/c2/T--UPO-Sevilla--glpFgel.png/324px-T--UPO-Sevilla--glpFgel.png)

Plate 130, 138, 127, 129 & 146.

[Back to the calendar](#calendar)

### **5th September**

**Microscopic simulation of biofilm growth model was executed in a supercomputer for two weeks to generate several 17000 bacteria biofilms.**

**Biofilm Module**

Inocula of KT2442-Tn7/128

**Glycerol Module**

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

**Migration to pSB1C3**

Inocula of the transformation of the fragments of 124, 125, 126, 135, 139, 145, 147 & 152 (four candidates each).

Ligation of framents of 123 and *glpF* with C3.

Ligation of fragment of 137 to pMRB1.

Inocula of plates with 130, 138, 127, 129 & 146.

[Back to the calendar](#calendar)

### **6th September**

**Biofilm Module**

Dilutions and prepare the INSTANT curves of KT2442-Tn7/128 for 30 h

**Glycerol Module**

1 inocula of KT2442 with pMRB172-*glpF* and other of KT2442 with Tn7 Ø in minimal media with glycerol as the carbon source

**Migration to pSB1C3**

Kit minipreps of the inocula of the plates with 130, 138, 127, 129 & 146.

Digestion of plasmids 130, 138, 127, 129 &146 with EcoRI-PstI.

Preparative electrophoresis gel and asolation of the inserts 130, 138, 127, 129 & 146.

Ligation of the inserts of 130, 138, 127, 129 & 146 with C3.

Manual miniprep of the inocula of the transformation of 124, 125, 126, 135, 139, 145, 147 & 152 with C3.

Diagnostic digestion with NotI to see the insert sizes.

[Back to the calendar](#calendar)

### **7th September**

**Biofilm Module**

Dye and measure the plates of KT2442-Tn7/128

**Glycerol Module**

Dilutions and preparation of the microtiter plates

**Migration to pSB1C3**

Transformation of the ligations of 130, 138, 127, 129 & 146 with C3.

Electrophoresis gel of the diagnostic digestion of the transformation of 124, 125, 126, 135, 139, 145, 147 & 152 with C3.

Second diagnostic digestion of the positives (124, 135, 152 and 125 were not positives, neither it was 126).

Electrophoresis gel with the second diagnostics digestion. Positives were 139, 145, 147 (parts BBa\_K1973014, BBa\_K1973016, BBa\_K1973011)

[Back to the calendar](#calendar)

### **8th 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 P*m* as it had a huge religation).

[Back to the calendar](#calendar)

### **9th September**

**Migration to pSB1C3**

Manual minipreps of all the inocula from yesterday.

Digestion of the inocula from the ligations with C3 with NotI to see the insert’s sizes.

Electrophoresis gel to see the result of the diagnostic digestion. 146, 127 & 130 were negatives.
123 was also negative.

[Back to the calendar](#calendar)

### **12th September**

**Glycerol Module**

Take out frozen bacteria with pSB1K3-*glpF8*

Obtaining of pMRB138 plasmid (pSB1K3-P*m*)

**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).

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### **13th September**

**Glycerol Module**

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

**Migration to pSB1C3**

Plate P*m*-*glpF*

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### **14th September**

**Glycerol Module**

Minipreps of the plasmids

Digestion of the plasmids (pSB1K3-P*m* with SpeI and PstI; pSB1K3-*glpF* with XbaI and PstI)

**Migration to pSB1C3**

Inocula of P*m*-*glpF*.

[Back to the calendar](#calendar)

### **15th September**

**Glycerol Module**

Electrophoresis of the digestions

Geneclean of *glpF*

Geneclean of the plasmid

**Migration to pSB1C3**

Kit miniprep of P*m*-*glpF* inocula.

Digestion of P*m*-*glpF* with EcoRI-PstI to extract the insert from the plasmid.

Preparative electrophoresis gel to isolate the insert.

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### **16th September**

**Migration to pSB1C3**

Arrival of IDT’s DNA concerning *lapC* and *nasR*.

Transformation of the DNA (leave the plates on the table for them to grow during the weekend).

[Back to the calendar](#calendar)

### **19th September**

**Glycerol Module**

Ligation of pSB1K3-P*m* and *glpF*

Obtaining of pMRB165 (Tn7-*nahR*-P*sal*-*PleD\**)

**Migration to pSB1C3**

Inocula of the transformations of IDT’s DNA from *lapC* and *nasR*, three candidates each.

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### **20th 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*.

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### **21st September**

**Glycerol Module**

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

Minipreps of pMRB165 and digestion with SpeI and PstI restriction enzymes

**Migration to pSB1C3**

lapC and *nasR* from the transformations.

glpF.

[Back to the calendar](#calendar)

### **22nd September**

**Glycerol Module**

Minipreps of the plasmids and diagnostic digestions

Electrophoresis. Positive results!!

Freeze bacteria containing pSB1K3-P*m*-*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 P*m*-*glpF*, *glpF*, *lapC*, *nasR* and 125 with pSB1C3.

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### **23rd September**

**Migration to pSB1C3**

Transformation of ligation of P*m*-*glpF*, 125, *glpF*, *lapC* & *nasR* with pSB1C3 in DH5α (leave the plates on the table for them growing during the weekend).

[Back to the calendar](#calendar)

### **26th September**

**Glycerol Module**

Ligation of pMRB165 and *glpF*

**Migration to pSB1C3**

Inocula of the transformations of the ligations (four candidates of each, P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*).
Plate pMRB120, 127, 140 & 146.

[Back to the calendar](#calendar)

### **27th September**

**Glycerol Module**

Transformation of the ligation

**Migration to pSB1C3**

Manual minipreps of yesterday’s inocula of P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*.

Diagnostic digestion of the ligations with NotI in order to see the fragment size of P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*.

Gel electrophoresis of the digestion.

Second diagnostic digestion of the positives of P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*.

Gel electrophoresis of the digestion.

Transformation of positive constructs of P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR* to freeze them and perform kit minipreps for submitting.

Inocula of pMRB120, 127, 140 & 146.

[Back to the calendar](#calendar)

### **28th 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 P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*.

Manual miniprep of 120, 127, 140 & 146.

Digestion of 120, 127, 140 & 146 with EcoRI and PstI.

Preparative electrophoresis gel and band asolation of 120, 127, 140 & 146 insert.

Ligation of 120, 127, 140 & 146 + C3.

Ligation of 121 + C3 (already have it stored in the freezer, cut with EcoRI-PstI from previous ligations).

[Back to the calendar](#calendar)

### **29th September**

**Glycerol Module**

Minipreps of the plasmids and diagnostic digestions

Electrophoresis. Positive results!!

Freeze bacteria containing Tn7-*nahR*-P*sal*-*PleD\**-*glpF* for storage

**Migration to pSB1C3**

Kit minipreps of yesterday’s inocula of postives of P*m*-*glpF*, *glpF*, 125, *lapC* & *nasR*.

Mesuring of DNA concentraton and dilution of it until 25 ng/ul for the submitting plate (parts BBa\_K1973035,BBa\_K1973026,BBa\_K1973003, BBa\_K1973036, BBa\_K1973037).

Transformation of the ligation of 120, 127, 140, 121 & 146 + C3.

[Back to the calendar](#calendar)

### **30th September**

[Back to the calendar](#calendar)

## **OCTOBER**

### **3rd October**

**Migration to pSB1C3**

Inocula of the transformation of the ligations of 120, 127, 140, 121 & 146 + C3.

[Back to the calendar](#calendar)

### **4th October**

**Migration to pSB1C3**

Manual minipreps of yesterday’s inocula of 120, 127, 140, 121 & 146 + C3.

Diagnostic digestion of the ligations with NotI in order to see the fragment size of 120, 127, 140, 121 & 146 + C3.

Gel electrophoresis of the digestion. 146 was again negative.

Second diagnostic digestion of the positives of 120, 127, 140 &121+ C3 (let it overnight).

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### **5th October**

**Migration to pSB1C3**

Gel electrophoresis of the digestion.

Transformation of positive constructs 120, 127, 140 & 121 + C3 to freeze them and perform kit minipreps for submitting (146 had no positives).

[Back to the calendar](#calendar)

### **6th October**

**Migration to pSB1C3**

Inocula from yesterday’s transformation (constructs 120, 127, 140 &121 + C3).

Plate all miniTn7 constructs for their minipreps made and DNA dilution performed (parts
BBa\_K1973006, BBa\_K1973009, BBa\_K1973010, BBa\_K1973012, BBa\_K1973019, BBa\_K1973021, BBa\_K1973022, BBa\_K1973023, BBa\_K1973024, BBa\_K1973025, BBa\_K1973027, BBa\_K1973028, BBa\_K1973029, BBa\_K1973031, BBa\_K1973032, BBa\_K1973038).

[Back to the calendar](#calendar)

### **7th October**

**Migration to pSB1C3**

Kit miniprep of the inocula performed yesterday.

Measuring of DNA concentration and dilution unit 25 ng/ul (parts BBa\_K1973033, BBa\_K1973001, BBa\_K1973015, BBa\_K1973000).

Parts BBa\_K1973031, BBa\_K1973032 did not grow. Made a mistake when plating them.

[Back to the calendar](#calendar)

### **10th October**

**Migration to pSB1C3**

Inocula of all miniTn7 devices for kit minipreps being performed on them.

Secong attempt of ligation of 123, 124, 130 and 135 with C3.

[Back to the calendar](#calendar)

### **11th October**

**Migration to pSB1C3**

Kit miniprep of all the inocula made yesterday.

Measure DNA concentration of all the miniTn7 devices and dilute them to 25 ng/ul.

Transformation of the ligations performed yesterday.

[Back to the calendar](#calendar)

### **12th October**

**Migration to pSB1C3**

Inocula of the transformatios performed yesterday (four candidates each construction).

[Back to the calendar](#calendar)

### **13th October**

**Migration to pSB1C3**

Kit minipreps of the inocula put yesterday.

Digestion of the minipreps with NotI to see the inserts sizes. All constructs had some positive when seen in the electrophoresis gel.

Second digestion performed to confirm the construcions.

Electrophoresis gel from the positives digested. All of them were positives.

As the minipreps where from the kit, measure of the DNA concentration and dilution until 25 ng/ul.

[Back to the calendar](#calendar)

### **14th October**

[Back to the calendar](#calendar)

### **17th October**

**Migration to pSB1C3**

Inocula of the transformation of P*m* with pMRB1 (six candidates).

Inocula of 146, 152, 126 and epimerase from the transformations of the ligations of them with C3 (four candidates each).

Plate Tn7-*nahR*-P*sal*-*glpF* and Tn7-*nahR*-P*sal*-*nasF*-*glpF*.

Measuring of DNA concentration of parts BBa\_K1973005, BBa\_K1973031, BBa\_K1973032, and dilution to 25 ng/ul.

Transformation of the DNA of positive from ligation of 122 with C3.

[Back to the calendar](#calendar)

### **18th October**

**Migration to pSB1C3**

Kit minipreps of the inocula from the ligations.

Diagnostic digestion with NotI to see insert sizes (146, 126, 152 and epimerase).

Diagnostic digestion with EcoRI-HindIII to see if P*m* ligation with pMRB1 gone well.

Electrophoresis gel to see the results of both digestions (126 and 152 were negative. We cannot send them to the registry, and the ligation with P*m* also was negative).

Second diagnostic digestion with EcoRI and EcoRV of the positives from first digestion.

Electrophoresis gel to see the results.

Dilution of the positives’ DNA until 25 ng/ul (parts BBa\_K1973030 and BBa\_K1973017).

Inocula of Tn7-*nahR*-P*sal*-*glpF*, Tn7-*nahR*-P*sal*-*nasF*-*glpF*, and 122.

[Back to the calendar](#calendar)

### **19th October**

**Migration to pSB1C3**

Kit miniprep of Tn7-*nahR*-P*sal*-*glpF*, Tn7-*nahR*-P*sal*-*nasF*-*glpF* and 122 inocula.

Measure of DNA concentration of these minipreps and dilution to 25 ng/ul.

**PREPARATION OF SUBMISSION PLATE**, and done the on-line form for the submissions.

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### **20th October**

**SUBMISSION OF THE PLATE**

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### **21st October**

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