iGEM 2018 — Team Rotterdam_HR

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# Team:Rotterdam HR/Notebook

# Notebook

# Notebook

As a team with only one computer scientist (and one electrical engineer who can program),
we wanted a notebook that automatically updates whenever someone adds something to our lab journal.
Luckily, we gained a sponsor that gave use an electronic lab journal as sponsorship.
With this, we can use their API to download specific sections from our lab journal and display them on or wiki.
But, as a multidisciplinary team we have multiple kinds of notebook entries: Software, Hardware and Wetlab.
Therefore we also used the Google Drive RESTful API for downloading software and hardware entries from google sheets files.

[More info about the notebook generator](https://2018.igem.org/Team%3ARotterdam_HR/Software)

[x] show wetlab entries
[x] show hardware entries
[ ] show software entries

[ ]

# Team page

### May 10

×

Rens Boeser

Created the first prototype for the team page

**01**

### Team page

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## May 10

[ ]

# Footer

### May 20

×

Rens Boeser

Social media created

**01**

### Footer

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## May 20

[ ]

# Footer

### May 21

×

Rens Boeser

Social media buttons same color and fixed iGEM style issues

**02**

### Footer

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## May 21

[ ]

# Notebook

### May 24

×

Rens Boeser

Multiple week summary styles created for the notes

**01**

### Notebook

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## May 24

[ ]

# Header

### May 27

×

Rens Boeser

Added the header with mobile compatibility als resized logos and readded them to the header

**01**

### Header

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## May 27

[ ]

# Main template

### May 28

×

Rens Boeser

Added template header and footer with 'under construction' content

**01**

### Main template

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## May 28

[ ]

# Header

### May 28

×

Rens Boeser

Small fixes and menu adjustments

**02**

### Header

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## May 28

[ ]

# Team

### May 29

×

Rens Boeser

Added a PI list with the secondary PI

**02**

### Team

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## May 29

[ ]

# Main page

### June 7

×

Rens Boeser

Designed and added content holders to the index Page

**01**

### Main page

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## June 7

[ ]

# Team

### June 9

×

Rens Boeser

Added myself to the teammember Page and did some bugfixing

**03**

### Team

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## June 9

[ ]

# Header

### June 9

×

Rens Boeser

Started working on a better looking menubar

**03**

### Header

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## June 9

[ ]

# Header

### June 10

×

Rens Boeser

Added the ability to add sub-menus to items in the menu bar. Integration into the website and added indication for which Page you are on

**04**

### Header

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## June 10

[ ]

# Construction pages

### June 13

×

Rens Boeser

Updated all Pages to the new style with an under construction sign

**01**

### Construction pages

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## June 13

[ ]

# Header

### June 14

×

Rens Boeser

Added a way to recognise Pages under construction using the menubar

**05**

### Header

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## June 14

[ ]

# Main page

### June 14

×

Rens Boeser

Updated the content containers so that they are responsive

**02**

### Main page

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## June 14

[ ]

# Architecture design [TC]

### June 18

×

Tom van Dijk | Mike Vrieswijk

The first draft of the architecture design for the temperature controller was made.

**01**

### Architecture design [TC]

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## June 18

[ ]

# Human Practices page

### June 22

×

Rens Boeser

Content added to the human practices page

**01**

### Human Practices page

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## June 22

[ ]

# Main page

### June 23

×

Rens Boeser

Content created and added to the main page

**03**

### Main page

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## June 23

[ ]

# Architecture design [TC]

### June 24

×

Tom van Dijk | Mike Vrieswijk

Today a few changes have been made to the architecture design of the temperature controller. The choice was made to use a premade power supply as opposed to a self-made one, because this was considering the time left to finish this device the best choice. A few alterations were made to make the architecture design better readable.

**02**

### Architecture design [TC]

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## June 24

[ ]

# First prototype [TC]

### June 25

×

Tom van Dijk | Mike Vrieswijk

A 3d drawing was made for the casing that will hold the temperature controller. The first prototype was printed, but was determent to be inadequate due to the lack of enough space for the cooler block.

**01**

### First prototype [TC]

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## June 25

[ ]

# Team

### June 26

×

Rens Boeser

Added the remaining instructors to the team

**04**

### Team

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## June 26

[ ]

# Second prototype [TC]

### June 26

×

Tom van Dijk | Mike Vrieswijk

In the drawing space was added to fit the cooler block for the temperature controller. Slits were added to the drawing for the nuts to slide into. The altered drawing was printed. The newly printed parts, cooler block, fan and Peltier element were assembled. Everything fit together.

**02**

### Second prototype [TC]

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## June 26

[ ]

# Assessing CooA Production

### June 26

×

Dustin van der Meulen

To start testing the production of CooA, we firstly resuspended and transformed the BioBricks from the kit witch we wanted to use (Table 1). This has been done using the iGEM Kit plate instructions (Protocol 1, step 1 - 3) and the NEB 10 béta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/μg pUC19. Inucbation was at 37°C.

Table 1 : Resuspended BioBricks from the iGEM 2018 Distribution kit and their uses.

|  |  |
| --- | --- |
| BBa\_K592009 | Blue Chromoprotein AmilCP |
| BBa\_B0031 | Ribosomal binding site (strong), derived from BBa\_B0030 |
| BBa\_B0032 | Ribosomal binding site (medium), derived from BBa\_B0030 |
| BBa\_B0030 | Ribosomal binding site (weak) |
| BBa\_B0015 | Double terminator (BBa\_B0010 & BBa\_B0012) |
| BBa\_J23100 | Strong Constitutive Anderson Promotor |
| BBa\_J23105 | Medium Constitutive Anderson Promotor |
| BBa\_J23113 | Weak Constitutive Anderson Promotor |
| BBa\_J45199 | Banana odor enzyme (ATF1) generator |
| BBa\_K1184000 | Killer Red |
| BBa\_K352001 | CooA |
| BBa\_K352011 | CooA responsive system |

After checking the plates on the 28th of June, we recieved the following results (Table 2):

Table 2 : Kolonies found after overnight growth.

|  |  |
| --- | --- |
| BBa\_K592009 | 20 colonies |
| BBa\_B0031 | 4 colonies |
| BBa\_B0032 | 5 colonies |
| BBa\_B0030 | 1 colonies |
| BBa\_B0015 | 6 colonies |
| BBa\_J23100 | 1 colonies |
| BBa\_J23105 | 98 colonies |
| BBa\_J23113 | 19 colonies |
| BBa\_J45199 | 0 colonies |
| BBa\_K1184000 | 4 colonies |
| BBa\_K352001 | 2 colonies |
| BBa\_K352011 | 4 colonies |

**01**

### Assessing CooA Production

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## June 26

[ ]

# Testrun [TC]

### June 27

×

Tom van Dijk | Mike Vrieswijk

A short test run was done with the newly assembled cooler. The test concluded that the temperature controller could cool a water droplet to the point of freezing and heat it back up to its boiling point.

**03**

### Testrun [TC]

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## June 27

[ ]

# Assessing CooA Production

### June 29

×

Dustin van der Meulen

After making a 20 mL overnight culture of our NEB 10 béta cells containing our BioBricks, we performed a mini prep (Protocol 3). Using our nanodrop spectrophotometer  which gave us the following DNA concentrations:

|  |  |
| --- | --- |
| BBa\_K592009 | 72,5 ng/μL |
| BBa\_B0031 | 118,9 ng/μL |
| BBa\_B0032 | 52,5 ng/μL |
| BBa\_B0030 | 488,6 ng/μL |
| BBa\_B0015 | 53,5 ng/μL |
| BBa\_J23100 | 148,3 ng/μL |
| BBa\_J23105 | 96,3 ng/μL |
| BBa\_J23113 | 151,8 ng/μL |
| BBa\_K118400 | 140,4 ng/μL |
| BBa\_K352001 | 77,7 ng/μL |
| BBa\_K352011 | 76,8 ng/μL |

After assessign our results, in the future we will perform the ethanol carry-over steps.

Also from the previously mentioned overnight culture 1 mL has been used to make Glycerol Stock (Protocol 4)

**02**

### Assessing CooA Production

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## June 29

[ ]

# Draft decision

### July 2

×

Tom van Dijk | Mike Vrieswijk

The choice to use a computer PSU as a power supply was made. The choice was made on the grounds, that computer PSU have a standard 12, 5 and 3.3V output. This is beneficial because these are the voltages necessary for the selected components.

**01**

### Draft decision

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## July 2

[ ]

# Assessing CooA Production

### July 2

×

Dustin van der Meulen

Today we performed a mini prep, digestion, dephosphorilation and ligation (following protocols 4 and 5) of a few promotor and RBS BioBricks. Because of administrative complications only the variants with J23113 and a RBS have been digested correctly.

**03**

### Assessing CooA Production

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## July 2

[ ]

# Assessing CooA Production

### July 2

×

Dustin van der Meulen

Today we transformed the previously made three combinations (BBa\_J23113 + BBa\_B0031 / B0032 / B0030) into chemically competent NEb 10 béta E.coli cells using the corresponding NEB protocol.

**04**

### Assessing CooA Production

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## July 2

[ ]

# Architecture design [TC]

### July 5

×

Tom van Dijk | Mike Vrieswijk

Changes were made to the architecture design of the temperature controller. Different voltage regulators were selected to fit the newly chosen power supply. To better readability of the architecture design a legend was added.

**03**

### Architecture design [TC]

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## July 5

[ ]

# Assessing CooA Production

### July 5

×

Dustin van der Meulen

After performing a mini prep and NotI digest on the 5th of July, and a gel electrophoresis today, we concluded that the cloning wasn't succesful.

**05**

### Assessing CooA Production

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## July 5

[ ]

# Electrical circuit design [TC]

### July 8

×

Tom van Dijk | Mike Vrieswijk

Custom footprints were made for multiple components and added to the parts library in the PCB design software.

**01**

### Electrical circuit design [TC]

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## July 8

[ ]

# Electrical circuit design [TC]

### July 9

×

Tom van Dijk | Mike Vrieswijk

The provisional electronic circuit design for the temperature controller was made.

**02**

### Electrical circuit design [TC]

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## July 9

[ ]

# Assessing CooA Production

### July 10

×

Dustin van der Meulen

Today we again performed a mini prep (overnight cultures from 10th of July) and digest. Sadly most digests did not give the expected results. Even though we had already progressed with the ligation. For now we've decided to progress with these ligation products.

**06**

### Assessing CooA Production

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## July 10

[ ]

# Electrical circuit design [TC]

### July 11

×

Tom van Dijk | Mike Vrieswijk

The electronic circuit design for the temperature controller was finished. Measuring point were added to the electronic circuit design.

**03**

### Electrical circuit design [TC]

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## July 11

[ ]

# Stock up

### July 11

×

Elise Grootscholten | Loraine Nelson | Paul Reusink

To have some basic biobricks ready, we stocked them by doing a plasmid isolation and freeze the DNA.

**01**

### Stock up

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## July 11

[ ]

# Basic parts

### July 11

×

Elise Grootscholten | Loraine Nelson | Paul Reusink

To start on a basic construct we did a digestion and ligation with the parts we isolated earlier. To prevent original constructs we did a defosphorylation after the digestion. We controlled the digestion by doing a gelelktroforeses.

**01**

### Basic parts

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## July 11

[ ]

# Board layout [TC]

### July 12

×

Tom van Dijk | Mike Vrieswijk

Started working on the board layout for the PCB of the temperature controller.

**01**

### Board layout [TC]

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## July 12

[ ]

# Basic parts

### July 12

×

Tom van Dijk | Randall de Waard

We continued with the ligated DNA by transformating it to Neb10Beta. This was plated onto agarplates and incubated for 1 day.

**02**

### Basic parts

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## July 12

[ ]

# Board layout [TC]

### July 13

×

Tom van Dijk | Mike Vrieswijk

Further work was done to the board layout for the temperature controller.

**02**

### Board layout [TC]

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## July 13

[ ]

# Basic parts

### July 13

×

Elise Grootscholten | Randall de Waard

The results of 7/12/2018 were collected, in action of this we decided to plate some ligated and transferred DNA from 7/12/2018 again. This we did because there wasn't a clear grow of colonies on the plates.

**03**

### Basic parts

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## July 13

[ ]

# Blue White screening on paper

### July 13

×

Elise Grootscholten | Randall de Waard

We tried to let bacteria grow on paper, therefore we used our own buisnesscards. We let the buisnesscards absorb some LB-medium and put them inside petridishes.

**01**

### Blue White screening on paper

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## July 13

[ ]

# Board layout [TC]

### July 15

×

Tom van Dijk | Mike Vrieswijk

Work has been done on the board layout for the temperature controller. The board layout is now finished.

**03**

### Board layout [TC]

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## July 15

[ ]

# Assessing CooA Production

### July 15

×

Dustin van der Meulen

After we transformed our ligation results from the 11th on the 12th, we recieved the following results on the 13th:

B0030+J23100: No growth
B0030+J23105: Unsure (small sports, maybe colonies)
B0031+J23100: Growth
B0031+J23105: Unsure (small sports, maybe colonies)
B0032+J23100: Growth
B0032+J23105: Unsure (small sports, maybe colonies)
K352001+B0015: Growth

On the 13th we retransformed the 4 ligations of which we didn't get conclusive results, which we looked at today:
B0030+J23100: No growth

B0030+J23105: Again unsure
B0031+J23105: No growth
B0032+J23105: No growth

After assessing the results we made overnight cultures of the following:

B0031+J23100

K352001+B0015

B0032+J23100

B0030+J23105 (1)

B0030+J23105 (2)

B0032+J23105

Also we retransformed the colonies we retransformed on the 13th again, and quickly spun our cells in a centrifuge, removing most of the supernatant and resuspending our cells before we spread them on Chloramphenicol plates.

**07**

### Assessing CooA Production

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## July 15

[ ]

# Basic parts

### July 16

×

Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard

To know if the ligation was succesfull we will controll the DNA, therefor we ented the bacteria's with DNA in LB-medium to grow overnight. We also plated the used bacteria's onto new plates to use for further experiments.

**04**

### Basic parts

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## July 16

[ ]

# Blue White screening on paper

### July 16

×

Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard

We did a blue white screening on our buisnesscards to see if bacteria had grew on them. We transferred the buisnesscards to new petrydishes and added ITPG and X-gal to the petrydishes.

**02**

### Blue White screening on paper

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## July 16

[ ]

# Notebook

### July 17

×

Rens Boeser

Frontend design for the notebook

**02**

### Notebook

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## July 17

[ ]

# Basic parts

### July 17

×

Rens Boeser | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

The resulst of 7/16/2018 showed that the DNA didnt ligate as planned. To be able to digest enough DNA we did a plasmid isolation again. We digested this DNA and froze it for further use.

**05**

### Basic parts

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## July 17

[ ]

# Stock up

### July 18

×

Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Mike Vrieswijk | Randall de Waard

We did another round of plasmid isolation for stocking.

**02**

### Stock up

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## July 18

[ ]

# Notebook

### July 19

×

Rens Boeser

Designed a way to follow experiments that take up multiple days and eventually designed a notebook entry page and made it a responsive page

**03**

### Notebook

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## July 19

[ ]

# Basic parts

### July 19

×

Dustin van der Meulen | Mei Ju Goemans| Randall de Waard

We digested all DNA we have in stock to test for our basic construct. We tested the DNA by doing a gelelktroforeses.

**06**

### Basic parts

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## July 19

[ ]

# Notebook

### July 21

×

Rens Boeser

Created a one page notebook. Made the notebook entry page more suited for the entry length

**04**

### Notebook

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## July 21

[ ]

# Assessing Gas Production BioBricks in E.Coli

### July 23

×

Elise Grootscholten | Randall de Waard

Transformations of the biobricks K133071, K173003 and I13453 are performed. K133071 will produce CO2 if there's pyruvate present and K173003 will produce CO2 if urea is present. I13453 is a promotor which will work if there's arabinose present.
The biobricks were transformed first into electrocometent cells and later into chemical competent cells from the strain NEb10Beta. After transformations the culture was plated on agar plates with antibiotics. The first transformations didn't work out, but the second did, because then the right competent cells were used.

**Experiment 1: Transformation biobricks**

Transformation of the biobricks K133071, K173003 and I13453 in NEB10Bèta.

**Materials**

- Biobricks (Bba\_K133071, Bba\_K173003 and Bba\_I3453)
- Nuclease free water
- LB- agar
- Variable Volume Pipettes
- Sterile pipetpoints
- Chemically competent cells ( NEB10Beta, 1x10^9)
- Heater (42°C)
- Incubator + shaker (37°C and 250 rpm)
- Eppendorf tubes (1,5 and 2 ml)
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium
-Chloramphenicol (40 mg/mL)

**Methods**
-K173003 --> pSB1C3 backbone, plate 6, well 15N
-K133071 --> pSB1C3 backbone, plate 6, wel 21A
-I13453 --> pSB1C3 backbone, plate 3, well 19O

To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24°C). After incubation the DNA is transferred to Eppendorf tubes and put on ice.

For the transformation 1 ul of the DNA was added to 50 ul chemically competent cells strain NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube in a heater (42°C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes.
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37°C and 250 rpm.

After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37°C.

**Results**
The first transformation was done in electrocompetent cells instead of chemocompetent cells so the first transformation did not succeed.

**Conclusion**

The second transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4°C and can be used for further experiments.
The CFU from the negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter again.

**01**

### Assessing Gas Production BioBricks in E.Coli

Show more

## July 23

[ ]

# ATP sensor

### July 24

×

Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

New biobricks, used to make an ATP sensor, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.

**01**

### ATP sensor

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## July 24

[ ]

# Gas output

### July 24

×

Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

New biobricks, which produces gasses, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.

**01**

### Gas output

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## July 24

[ ]

# Notebook generator

### July 24

×

Rens Boeser

Started working on an automatic notebook creator

**01**

### Notebook generator

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## July 24

[ ]

# Assessing Gas Production BioBricks in E.Coli

### July 24

×

Elise Grootscholten | Randall de Waard

To be able to always get the necessary biobricks, there have been made glycerolstocks of the transformed biobricks K133071, K173003 and I13453. The glycerolstocks are stored at -80 degrees Celsius. If needed, they can be retrieved from this storage to use for experiments.

**Experiment 2: Glycerolstocks**

Making glycerol stocks of the biobricks K133071, K173003, I13453.

**Materials**
- 87% glycerol
- culture with biobrick plasmids
- LB-medium
- Variable Volume Pipette
- Sterile pipetpoints

**Methods**
The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was incubated for a night at 37°C.

The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80°C.

The stocks were stored at -80°C in drawer 3, tower 1, drawer

**02**

### Assessing Gas Production BioBricks in E.Coli

Show more

## July 24

[ ]

# Assessing different ATP sensors

### July 24

×

Loraine Nelson | Elise Grootscholten | Paul Reusink

Today we did a transformation with new biobricks. The biobricks were built into chemically competent cells from the strain NEb10Beta. After transformation the culture was plated on agar plates with antibiotics. The results were checked the next day.

Materials:

- Biobricks (Bba-K1499004 and Bba- K1614019)
- Nuclease free water
- LB- agar
- Variable Volume Pipettes
- Sterile pipetpoints
- Chemically competent cells ( NEB10Beta, 1x10^9)
- Heater (42°C)
- Incubator + shaker (37°C and 250 rpm)
- Eppendorf tubes (1,5 and 2 ml)
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium

Method:

To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24°C). After incubation the DNA is transferred to Eppendorf tubes and put on ice.

For the transformation 1 ul of the DNA was added to 50 ul chemically competent cells strain NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube in a heater (42°C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes.
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37°C and 250 rpm.

After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37°C.

Results:
Tabel 1 shows the results of the plates which were incubated.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Biobricks: | Antibiotics: | Dilution: | Colonies: | CFU: |
| Bba-K1614019 | Can | 0x | 109 | 1\*10^3 /ml |
| Bba-K1614019 | Can | Centrifuged | >300 | - |
| Bba-K1499004 | Can | 0x | 33 | 3\*10^2 /ml |
| Bba-K1499004 | Can | Centrifuged | 228 | - |
| Positive controle: Bba- K1499004 | None | 0x | > 300 | - |
| Negative controle: outgrow medium | none | 0x | 18 | - |

Tabel 1: results from the transformation with the biobricks Bba-K1614019 and Bba-K1499004.

The plates show CFU which can be used for further experiments.  The negative controle also has CFU, this shows that the outgrow medium was infected and not sterile.
The plates are stored by 4°C.

Conclusion:

The transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4°C and can be used for further experiments.
The CFU from the negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter again.

**01**

### Assessing different ATP sensors

Show more

## July 24

[ ]

# Notebook generator

### July 25

×

Rens Boeser

Automatic generation of entries completed

**02**

### Notebook generator

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## July 25

[ ]

# Google Drive API

### July 25

×

Rens Boeser

Used the Google drive REST API to download the entries from drive

**01**

### Google Drive API

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## July 25

[ ]

# Automatic uploader

### July 25

×

Rens Boeser

Used the igemwiki API to update pages using software

**01**

### Automatic uploader

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## July 25

[ ]

# Assessing Gas Production BioBricks in E.Coli

### July 27

×

Elise Grootscholten | Randall de Waard

For further experiments there is isolated DNA needed of the biobricks J23100, K133071, K173003 and I134353. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments. J23100 (from the glycerolstock): 325,59 ng/ul, K173003: 217,06 ng/ul, K133071: 186,79 ng/ul, I13453: 88,18 ng/ul

**Experiment 3: Minipreps (DNA isolation)**
27-07-18

Minipreps of the following biobricks in NEB10Bèta:
-J23100: Constitutive promoter
-K133071: Urea --> ammonia + CO2
-K173003: Pyruvate --> acetaldehyde + CO2
-I134353: Promoter (AraC protein binds with arabinose)

**Materials**
- Mini prep, Plasmid Purification Kit, Machery Nagel
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

**Methods**
**Miniprep protocol Plasmid Purification Kit, Machery Nagel**
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at 37°C and 150 rpm. The culture was mini prepped the next day. For isolation about 6 mL overnight culture was used.

**Plasmid DNA purification** (**NucleoSpin� Plasmid EasyPure)
Machery Nagel Mini preps**

**1 Cultivate**

**and harvest bacterial cells**

**12,000 x g, 30 s**

**2 Cell lysis 150 μL Buffer A1**

**250 μL Buffer A2**

**RT, up to 2 min**

**350 μL Buffer A3**

**3 Clarification of the lysate**

**> 12,000 x g, 3 min**

**4 Bind DNA**

**Load supernatant**

**1,000�2,000 x g, 30 s**

**5 Wash and dry silica**

**membrane**

**450 μL Buffer AQ**

**> 12,000 x g, 1 min**

**6 Elute DNA 50 μL Buffer AE**

**RT, 1 min**

**> 12,000 x g, 1 min**

**Results**
J23100: 325,59 ng/ul
K173003: 217,06 ng/ul
K133071: 186,79 ng/ul
I13453: 88,18 ng/ul

Figure 1: Minipreps 27-07-18

**03**

### Assessing Gas Production BioBricks in E.Coli

Show more

## July 27

[ ]

# Assessing different ATP sensors

### July 27

×

Loraine Nelson | Elise Grootscholten | Paul Reusink

To be able to always get the necessary biobricks, we made an glycerol stock after every transformation. The glycerol stocks are stored at -80 degrees Celsius. If needed, they can retrieved from this storage to use for experiments.

Making glycerol stocks of the biobricks.

Materials:
- 87% glycerol
- culture with biobrick plasmids
- LB-medium
- Variable Volume Pipette
- Sterile pipetpoints

Method:

The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was incubated for a night at 37°C.

The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80°C.

The stocks were stored at -80°C in drawer 3, tower 1, drawer

**02**

### Assessing different ATP sensors

Show more

## July 27

[ ]

# Automation program

### July 29

×

Rens Boeser

Connected the page uploader to the notebook generator

**01**

### Automation program

Show more

## July 29

[ ]

# Automatic uploader

### July 29

×

Rens Boeser

Created a program that uploads all updated pages

**02**

### Automatic uploader

Show more

## July 29

[ ]

# Assessing CooA Production

### July 29

×

Dustin van der Meulen

After considering multiple tactics, we've decided to follow the standard iGEM 3A assembly in our following cloning steps. To this end we started with an overnight digest of all the different parts.

|  |  |  |
| --- | --- | --- |
| BioBrick BBa\_ code | Restriction enzyme 1 | Restriction enzyme 2 |
| J23100 | EcoRI | SpeI |
| J23105 | EcoRI | SpeI |
| J23113 | EcoRI | SpeI |
| B0030 | EcoRI | XbaI |
| B0031 | EcoRI | XbaI |
| B0032 | EcoRI | XbaI |
| K352001 | SpeI | PstI |
| B0015 | XbaI | PstI |

Used pipette scheme from protocol 7 to prepare the DNA to be digested with multiple combinations of EcoRI-HF, SpeI-HF, PstI and XbaI.

With this we started a digest of the following BioBricks using the following Restriction enzyme couples:

|  |  |  |
| --- | --- | --- |
| BioBrick BBa\_ code | Restriction enzyme 1 | Restriction enzyme 2 |
| J23100 | EcoRI | SpeI |
| J23105 | EcoRI | SpeI |
| J23113 | EcoRI | SpeI |
| B0030 | EcoRI | XbaI |
| B0031 | EcoRI | XbaI |
| B0032 | EcoRI | XbaI |
| K352001 | SpeI | PstI |
| B0015 | XbaI | PstI |

Digestion was started on the 30th of July, ~16:00, 37°C and 185 RPM.
Digestion was stopped on the 31st of July, ~10:00.

**09**

### Assessing CooA Production

Show more

## July 29

[ ]

# Making competent NEB10beta cells

### July 30

×

Loraine Nelson | Jos Veldscholte

Made stock solution of MgSO4 and pure culture of NEB10 beta cells

**Making 1 M sterile MgSO4**
≈ 14.00: Dissolved 12,32 g MgSO4 in 50 mL demi water en filtered through 0.22 μm filter.

**Making pure culture of NEB10 beta cells**
≈15.00:20 mL LB agar + 10 μL tetracyclin (50 mg/mL) to pour 1 plate.
16:30: streaked bacteria from glycerol stock: -80, section III, tower 1, shelf IV, box III, 6B
Left in 37°C room for o.n. growth.

**01**

### Making competent NEB10beta cells

Show more

## July 30

[ ]

# Page generator

### July 31

×

Rens Boeser

Built a page builder that automatically adds the header and footer before uploading

**01**

### Page generator

Show more

## July 31

[ ]

# Google Drive API

### July 31

×

Rens Boeser

Redone the google API handler for reusability and readability and made the google API handler generic

**02**

### Google Drive API

Show more

## July 31

[ ]

# Automation program

### July 31

×

Rens Boeser

revamped the main program

**02**

### Automation program

Show more

## July 31

[ ]

# Assessing CooA Production

### July 31

×

Dustin van der Meulen

Though we had started with our ligation using the rAPId dephos and ligation kit, we'd simultaniously put our DNA on gel, later revealing the digest to not have been succesful. Carefull examination of our work made us realise the used restriction enzyme dilutions to not have been made correctly.

Using the rAPid Dephos and Ligation Kit we performed a dephosphorilation and ligation of our DNA.
After checking our gel with our digestion products we later determined that the digestion didn't go as expected

**10**

### Assessing CooA Production

Show more

## July 31

[ ]

# Making competent NEB10beta cells

### July 31

×

Loraine Nelson | Jos Veldscholte

Made Ksi broth (for competent cells) and started o.n. cultures

**Make Ksi broth (for competent cells)**
Dissolved 20 g Tryptone (Lab M limited) and 5 g Yeast extract (Lab M limited) in 1 liter demi water, added 10 ml 1 M NaCl and adjusted pH to 7.5 with KOH.
Autoclaved.
16:30
2 tubes with 2.5 mL Ksi inoculated with 1 colony NEB10beta from yesterday's plate. (Added 50 microliters of 1 M MgSO4 at 16:30 becauce I forgot to ad it to de autoclaved medium.
Added 10 mL of 1 M MgSO4 to each half liter autoclaved medium (=Ksi).

**02**

### Making competent NEB10beta cells

Show more

## July 31

[ ]

# Automation program

### August 1

×

Rens Boeser

Finished redoing the application for generating and uploading pages

**03**

### Automation program

Show more

## August 1

[ ]

# Making competent NEB10beta cells

### August 1

×

Loraine Nelson | Jos Veldscholte

Made large NEB10 beta culture and aliquoted in 50 microliter portions and snap froze in dry ice/isopropanol

**Making competent NEB10 beta cells**
8:45:
Both cultures were fine.
Transfered one culture completely to 100 ml Erlenmeyer with 25 mL Ksi (RT). In shaking incubator at 37° C at 200 rpm.
Filled three one liter Erlenmeyers with 175 mL Ksi and put in shaking incubator in 37° C room to adjust temperature.
25 ml culture: at 8:40 OD600 was 0.64.
Transferred 8 ml to each 175 mL Erlenmeyer. Incubate at  37° C shaking rigorously (no speed indicator).
9:40: OD600 = 0.-9
10:30: OD600 = 0.30
10:50 OD600 = 0.46
10:58 OD600 = 0.55
Tranferred all cultures to 10 50 mL tubes and tare in pairs.
On ice 11:15
11:35: in Sorvall 4500 rpm, 15 min 0° C.
After the run, fluid was on the rotor and in the rotor holes.
Cleaned with lab desinfectant. Rinsed with water and dried with tissue.
From here on everything in the cold room with tubes on ice:
Resuspended pellets in 16,6 ml ice cold TFB1 (protocol "competente-cellen-maken"l) each with 10 mL plastic pipet and pipetboy.
Combined into four 50 mL tubes. Centrifuged 15 min at 4500 rpm.
Resuspended pellets in 60 mL TFB1, kept on ice for 20 min and centrifuged 15 min 4500 rpm.
Respuspended pellets in 10,5 ml TFB2 (protocol "competente-cellen-maken"l). Left on Ice for 10 min.
Aliquotted into 50 μL portions in 2 mL conical tubes and snap froze in isopropanol on dry ice.
Transferred to -80°C Freezer.

**03**

### Making competent NEB10beta cells

Show more

## August 1

[ ]

# Notebook

### August 2

×

Rens Boeser

Added month categorization to the page

**05**

### Notebook

Show more

## August 2

[ ]

# Assessing CooA Production

### August 2

×

Dustin van der Meulen

We've redone the work mentioned in the previous CooA entry, though today our gel electrophoresis revealed we were only partially succesful, as only the digests with EcoRI and SpeI seem to be correct.

Experiment 9 and 10 have been repeated with a new enzyme dilution.
7th of august: review of the digestions on gel reveal that only the digestions with EcoRI-HF and SpeI-HF worked, while the PstI and XbaI digestion did not retrieve the expected results

**11**

### Assessing CooA Production

Show more

## August 2

[ ]

# Making competent NEB10beta cells

### August 2

×

Loraine Nelson | Jos Veldscholte

Tested competence by transformation of pUC19

Test sample from NEB competent cells contains 50 pg pUC19 per μL.
Used 2 μL per transformation (= 100 ng).
Took three tubes from one of the two boxes.
A 100 ng pUC19
B 100 ng pUC19
C nothing
30 min on ice (11:00)
A 30 s heatshock in waterbath (11:30)
B 90 s heatshock in waterbath (11:30)
C was kept on ice still.
5 min on ice.
Added 1 ml of LB (11:40)
60 min shaking in 37 °C room (shaking slowly)

Poured LB agar plates with 50μg/ml Ampicillin and one plate without antibiotic.

12:40
Added 1 μL of culture A or B to 99 μL LB (A4 and B4)
Added 10 μL of culture A or B to 90 μL LB (A3 and C3)
Plated A3 and 4 and C3 and 4 on LB agar amp.
Plated 100 μL A, B and C (A2, B2 and C2) on LB agar amp.
Plated 100 μL C on plate without antibiotics (to test whether cells are alive)
Centrifuged A and B 6000 rpm in microcentrifuge for 2 min.
Aspirated supernatant.
Vortexed bacteria in remainder of the medium (>100 μL)
Plated on LB agar amp. plates (A1 and B1).

Consequently:
A was heat shocked for 30 s.
B was heat shocked for 90 s.
1 = 90% of the transformation, 2 = 10%, 3 = 1% and 4 = 0,1%

Put in automated incubator. Not sure about whether the sytem wil work. Is it on?
Colonies to be counted next monday by Dustin.
If there are no colonies visible, please extend incubation time!

**04**

### Making competent NEB10beta cells

Show more

## August 2

[ ]

# Making competent NEB10beta cells

### August 3

×

Loraine Nelson | Jos Veldscholte

Counted colonies. Result: no transformation.

Colonies where counted at ~10:00. Only plate A1 showed signs of colonies (~15). All the others where empty.
After leaving the cells in the incubator for an extra half a day, there where no new colonies.

**05**

### Making competent NEB10beta cells

Show more

## August 3

[ ]

# Assessing CooA Production

### August 4

×

Dustin van der Meulen

The previous two CooA entries have been repeated, though this time we remade the PstI and XbaI enzyme dilutions. This time the digests where as expected.

Experiment 11 has been repeated, but with new dilutions of PstI and XbaI.
After the incubation it was noted that the cup containging BBa\_B0015 was almost empty, so it will have to be digested again. (did not happen) --> in the end we had enough

13th of august --> dephos and ligation was peformed on the obtained biobrick combinations following protocol 8

**12**

### Assessing CooA Production

Show more

## August 4

[ ]

# Making competent NEB10beta cells

### August 7

×

Loraine Nelson | Jos Veldscholte

Repeated transformation.

Part 4 has been repeated, repeating the same steps but adding two extra positive controlls.

**06**

### Making competent NEB10beta cells

Show more

## August 7

[ ]

# Assessing different ATP sensors

### August 9

×

Loraine Nelson | Elise Grootscholten | Paul Reusink

For further experiments we needed isolated DNA. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.

Materials:
- Mini prep, Plasmid Purification Kit, Machery Michels
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points

Method:
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at 37°C and 150 rpm. The culture was mini prepped the next day. For isolation, 5,4 ml was added step wise in a 2ml Eppendorf cup. The culture was then centrifuged at >12000x g for 30 seconds. The supernatant was deposed, and the pellet was resupended with A1 buffer and vortexed. To the resuspended culture was then.

**03**

### Assessing different ATP sensors

Show more

## August 9

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 10

×

Elise Grootscholten | Randall de Waard

The biobricks J23100, K133071, K173003 and I134353 were sucessfully digested after the second time. After the digestions the biobricks K133071 and K173003 were dephosphorylated and ligated with the inserts J23100 and I13453. This was done in the original backbone of K133071 and K173003, and not another control backbone. To know if the biobricks were right ligated this was done by testing practically. See the experiments: *Testing gas production.*

**Experiment 4: Digestion, Defosforylation and ligation**
**Digestions**
30-07-18 and 08-08-18

Biobricks: J23100, K133071, K173003 and I13453

Used pipette scheme (Table 1) to prepare the DNA to be cut with multiple combinations of EcoRI-HF, SpeI-HF, PstI and XbaI.

|  |  |
| --- | --- |
| DNA | ~ 2 μg |
| NEBbuffer 2.1 | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 2 μL |
| Restriction enzyme 2 (1 U/μL) | 2 μL |
| Nuclease free water | Fill to 100 μL |

*(Table 1 ; Double Digest Pipette Scheme using : <https://nebcloner.neb.com/#!/redigest>, and doubling the amount)*

**Biobricks:**
J23100: Constitutive promotor
K133071: Urea --> ammonia + CO2
K173003: Pyruvate --> acetaldehyde + CO2
I134353: Promotor (AraC protein binds with arabinose)

With this we started a digest of the following BioBricks using the following Restriction enzyme couples (Table 2)

|  |  |  |  |
| --- | --- | --- | --- |
| **Number** | **BioBrick BBa\_ code** | **Restriction enzyme 1** | **Restriction enzyme 2** |
| 9 | J23100 (35 bp) | EcoRI | SpeI |
| 11 | K133071 (1707 bp) | EcoRI | XbaI |
| 12 | K173003 (3052 bp) | EcoRI | XbaI |
| 10 | I13453 (130 bp) | EcoRI | SpeI |

*Table 2: Biobricks and restriction enzymes*

Digestion was started on the 30th of July, ~16:00, 37°C and 185 RPM.
Digestion was stopped on the 31st of July, ~10:00.

This has been done in tandem with the corresponding part from [Cloning Mulitple BioBricks to Assess CooA Production](/members/experiments/browser/#view=experiment&nodeID=313406) Part X

**Dephosphorylation**
10-08-18

K133071 and K173003 were dephosphoralized:

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 25 μL | Vector DNA (0,5 μg) |
| 3 μL | phosphatase buffer (10x) |
| 1 μL | milli Q |
| 1 μL | phosphatase (1 U/μL) |
| 30 μL | Total |

*Table 3: Pipetting scheme*

The new DNA concentration of the reaction mix is 0,5 μg/30 μL
The reaction mix was incubated for 10 minutes at 37 °C
The phosphatase was inactivated for 2 minutes at 75 °C

For the ligation mix 100 ng of vector DNA was used from the dephosphorylation reaction mix.
The following vector-insert combinations were ligated:

|  |  |  |
| --- | --- | --- |
| **Vector** | **Insert 1** | **Insert 2** |
| K133071 | J23100 | I13453 |
| K173003 | J23100 | I13453 |

*Table 4: Biobrick combination*

The original plasmids of the vector and inserts that were digested are about the same size in bp and the ratio vector DNA-insert DNA is determined to be 1:3. Therefore, the amount of insert DNA that was used for the ligation was 140 ng (rounded up to 7 μL of the digestion reaction mix). Because there was only 5 μL left of the K352001 insert DNA, the total 5 μL were used and an extra 2 μL of milli Q water were added to the pre-ligation mix.

Pre-Ligationmix:

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 6 μL | Vector DNA (0,5 μg/30 μL) |
| 7 μL | Insert DNA (2 μg/100 μL) |
| 4 μL | Delutionbuffer (5x) |
| 3 μL | Milli Q |
| 20 μL | Total |

*Table 5: Pre-ligationmix pipette scheme*

Ligationmix:
incubation at room-temperature for 20 minutes.

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 10 μL | Pre-ligationmix |
| 2 μL | 10x T4 ligationbuffer |
| 1 μL | T4 DNA ligase (1U/μL) |
| 8 μL | Milli Q |
| 20 μL | Total |

*Table 6: Ligationmix pipette scheme*

All the vectors contain the CamR gene. 30 plates were poured with Cam (14 for the transformed cells (100μL), 14 for the transformed cells (900 μL centrifuged and resuspended in 100 μL and 1 positive and 1 negative control)

**04**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 10

[ ]

# Urea and sodium pyruvate test for resistance E.coli

### August 10

×

Randall de Waard | Elise Grootscholten

Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.

**Experiment 1: Testing different concentrations of Urea and Sodium pyruvate**
**08-08-18**

Testing different amountsof urea and sodiumpyruvate to know which concentrations the bacteria survive.

-First let the LB agarose heat up (95 degrees Celcius)
-Second, weigh urea and sodium pyruvate in beakers (see table 1)

|  |  |  |
| --- | --- | --- |
| **What** | **How much** | **Solution** |
| Urea | 1 g | 1 mL purified water |
| Urea | 2,4 g | 2 mL purified water |
| Urea | 4,8 g | 4 mL purified water |
| Sodium pyruvate | 5,7 mL | None |

*Table 1: weighing urea and sodium pyruvate for LB agarose plates.*
-Purify these substances, so it is free of bacteria. Work beneath the flame and pour it in to plastic 50 mL tubes.
-Pour the LB agarose in the plastic tubes when it's about 45 degrees Celcius and mix gently.
-Pour the LB agarose mix in a petridish and wait until it's dry. Do this for all the plastic tubes.
-Also fill 2 petridishes with LB agarose without other substances (positive and negative control).
-prick a BL21(DE3) bacteria and put it in a 100 mL erlenmeyer with 20 mL culture medium.

**09-08-18**

Pipette 100 ul overnight culture on each plate, except on the negative control, and divide it. (24h 37 degrees Celcius)

**Results**

|  |  |  |  |
| --- | --- | --- | --- |
| **What** | **How much** | **Solution** | **Result** |
| Urea | 1 g | 1 mL purified water | Grow |
| Urea | 2,4 g | 2 mL purified water | No grow |
| Urea | 4,8 g | 4 mL purified water | No grow |
| Sodium pyruvate | 5,7 mL | - | Grow |
| Positive control | - | - | Grow |
| Negative control | - | - | No grow |

*Table 1: Results experiment 1*

**Conclusion**

More tests will be done with urea and sodiumpyruvate because with 1 g urea the bacteria live and with 2,4 g urea the bacteria die.
The bacteria survive 5,7 mL sodiumpyruvate so we will test with higher concentrations.

**01**

### Urea and sodium pyruvate test for resistance E.coli

Show more

## August 10

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 13

×

Elise Grootscholten | Randall de Waard

Transformations of the biobricks (K133071 + J23100), (K13071 + I1345), (K173003 + J23100) and (K173003 + I13453) in NEB10Bèta. There was no grow except for the biobrick combination K133071 + J23100.

**Experiment 5: Transformation**

13-08-2018
Transformations of the ligations of experiment 4

**Materials**

-Chloramphenicol (40 mg/mL)
-LB-agar
-Ligations
-Heat block,42 degrees Celcius
-Shaker, 37 degrees Celsius
-Chemo competent cells NEB10bèta (New England Biolabs)

**Methods**

10 ul ligationmix is used for every transformation

# Transformation Protocol Overview

Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs.

## Protocol

1. Thaw competent cells on ice.
2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3. Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4�5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds\*. Do not mix.
6. Add 950 μl of room temperature media\* to the tube.
7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Spread 50�100 μl of the cells and ligation mixture onto the plates.
10. Incubate overnight at 37°C.

    \* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells� manufacturer.

Instead of 2 ul of the ligation mix, use 10 ul ligation mix.

**05**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 13

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 14

×

Elise Grootscholten | Randall de Waard

A colony PCR is done for the NEB10Bèta E.coli cells with expected the biobrick combination of K133071 with J23100. Nevertheless, on a gel the difference with and without promotor couldn't be seen. So there must be another way of proving the right biobricks are there.

**Expermiment 6: Colony PCR K133071 + J23100**
14-08-18

Mastermix

|  |  |  |
| --- | --- | --- |
| **What** | **1 reaction** | **30 reactions** |
| Taq buffer(10x) | 5 ul | 150 ul |
| 10 mM dntp | 1 ul | 30 ul |
| 10 uM Forward primer | 1 ul | 30 ul |
| 10 uM Reverse primer | 1 ul | 30 ul |
| Taq polymerase | 0,25 ul | 7,5 ul |
| Nucease free hydrogen | 41,75 ul | 1252,5 ul |
| **Total** | **50 ul** | **1500 ul** |

**06**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 14

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 15

×

Elise Grootscholten | Randall de Waard

The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 have been performed again. There was a lot of grow on the agar plates. To know if the biobricks were right ligated this was done by testing practically. See the experiments: *Testing gas production.*

**Experiment 7: Ligation of biobricks**
15-08-18

K173003 + J23100
K173003 + I13453
K133071 + I13453

| Volume | Compound |
| --- | --- |
| 6 μL | vector DNA (0,5 μg/30 μL) (100 ng) |
| 14 μL | insert DNA (2 μg/100 μL) (280 ng) |
| 4 μL | dilutionbuffer (5x) |
| 1 μL | milli Q water |
| 25 μL | Total |

Use 10 mL

Ligationmix:

| Volume | Compound |
| --- | --- |
| 10 μL | pre-ligationmix |
| 2 μL | 10x T4 ligationbuffer |
| 1 μL | T4 DNA ligase (1U/μL) |
| 8 μL | milli Q water |
| 20 μL | Total |

incubation at room-temperature for 20 minutes.
Store at -20 degrees Celcius

**07**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 15

[ ]

# Assessing CooA Production

### August 16

×

Dustin van der Meulen

We've performed a colony PCR on 15 random (though not the red/purple -esque colonies, which are collored only because they contain BBa\_J04450) colonies from J23105+B0032 on the 13th. We also ligated the following biobricks anew:
B0031 + J23105
B0031 + J23113
B0032 + J23100
B0032 + J23105

We also transformed these biobricks into *E.coli* on the 16th, prepping and digesting them later on, though these and the PCR did not give us the result we'd hoped for.

Today a colony PCR will be performed to check our results from last friday, namely the transformation product of BBa\_B0032 en BBa\_J23105:

Mastermix

|  |  |  |
| --- | --- | --- |
| **What** | **1 reaction** | **30 reactions** |
| Taq buffer(10x) | 5 ul | 150 ul |
| 10 mM dntp | 1 ul | 30 ul |
| 10 uM Forward primer | 1 ul | 30 ul |
| 10 uM Reverse primer | 1 ul | 30 ul |
| Taq polymerase | 0,25 ul | 7,5 ul |
| Nucease free hydrogen | 41,75 ul | 1252,5 ul |
| **Total** | **50 ul** | **1500 ul** |

15 colonies have been randomly selected to be checked via PCR.

**13**

### Assessing CooA Production

Show more

## August 16

[ ]

# Testing gas production

### August 16

×

Elise Grootscholten | Paul Reusink

Making a set up for the gas production testing and testing it with NEB10Bèta with the expected biobricks in it (K133071 + J23100) and a negative control.

**Experiment 1: Gasproduction testing**
15-08-18
Making a set up for the gas production testing and testing it with NEB10Bèta with the expected biobricks in it (K133071 + J23100) and a negative control.

**Materials**
-15 mL tubes
-Little glass tubes
-Cocktail prickers
-Culture medium
-Urea
-Chloramphenicol (40 mg/mL)

**Methods**
Stick tape on the little tube and cover the little tubes with tin foil. This can now enter the autoclave.
Make overnight cultures (20 mL)of the desired colonies in culture medium with 17,5 ul/ 20mL Chloramphenicol.

After a day, prepare the culture medium for the tests.
Put 1 g urea and 17,5 ul Chloramphenicol in 20 mL culture medium.

Pipette 10 mL of the overnight culture in the 15 mL tube en centrifuge 5000 rpm fot 5 minutes. Throw the supernatant away.
Fill the 15 mL tube and the little tube to the edge with culture medium. Work sterile.
Put a paper on the little tube and turn it around.
Then hold it above the 15 mL tube and pull the paper away and let the little tube fall into the 15 mL tube.
Try to do this so there's no gas in the little tube.
Now wait until gas is produced.

16-08-18
Making a set up for the gas production testing and testing it with NEB10Bèta with the expected biobricks in it (K133071 + J23100).
20 different colonies are tested.

**Materials**
Use the same materials as above.

**Methods**
Use the protocol above.
Make 700 mL culture medium with 35 g urea and 612,5 ul Chloramphenicol (40 mg/mL). This cannot be done sterile.
The culture medium can be used for the 20 different colonies with expected biobricks: K133071 + J23100.

**Results**
Gas was let into the little tubes so it was difficult to see whether there was gasproduction or not. Mainly in the negative control the tubes started with gas in it.

Figure 1: Little tube with cocktailpricker and 15 mL tube

Figure 2: Set up testing gas production

Figure 3. tube 1,2,3 and 4 starting amounts of air

Figure 4: After the weekend tube 1, 2, 3 and 4

Figure 5. tube 5,6,7 and 8 starting amounts of air

Figure 6: After the weekend tube 5, 6, 7 and 8

Figure 7. tube 9 and 10 strating amounts of air

Figure 8: After the weekend tube 9 and 10

Figure 9. tube 11,12,13,and 14 starting amounts of air

Figure 10: After the weekend tube 11, 12, 13 and 14

Figure 11. tube 15,16,17 and 18 starting amounts of air

Figure 12: After the weekend tube 15, 16, 17 and 18

Figure 13. tube 19 and 20 starting amounts of air

Figure 14: After the weekend tube 19 and 20

**01**

### Testing gas production

Show more

## August 16

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 21

×

Elise Grootscholten | Randall de Waard

Minipreps are made of colonies 11 and 19 of biobrick combination K133071 + J23100. Results: 11. 270,57 ng/ul 19. 253,59 ng/ul.

**Minipreps**
21-08-18

Minipreps of colonies 11 and 19 (see Testing gasproduction experiment 1)

**Materials**
- Mini prep, Plasmid Purification Kit, Machery Michels
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

**Methods**
**Miniprep protocol Plasmid Purification Kit, Machery Nagel**
See experiment 3

**Results**

11. K133071 + J23100: 270,57 ng/ul
19. K133071 + J23100: 253,59 ng/ul

Figure 1: Results minipreps colonies 11 and 19

**08**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 21

[ ]

# Testing gas production

### August 22

×

Elise Grootscholten | Paul Reusink

Making a set up for the gas production testing and testing it with NEB10Bèta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453) and a negative control. Colonies 9 and 10 are chosen use for further tests because there was the biggest amount of gas production.

**Experiment 2: Gasproduction testing**
**20-08-18**

Making a set up for the gas production testing and testing it with NEB10Bèta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453) and a negative control

**Materials**
See materials Experiment 1

**Methods**
See methods Experiment 1
850 mL culture medium is made with 21,5 g LB for 42 erlenmeyers (also for CooA production).
Overnight cultures are made of 10 colonies each of K173003 + I13453 and K173003 + J23100.

**21-08-18**

K173003 + J23100 are tested (see methods experiment 1)
Overnight cultures are made of  K133071 + I13453 and K173003 + I13453 are tested later.

**Results**
Colonies 9 and 10 are chosen use for further tests.

Figure 1. Starting amounts of air in tube 1,2,3 and 4.

Figure 2. Gas formation in tube 1,2,3 and 4. 30 minutes after adding
Sodium pyruvate.

Figure 3. Starting amounts of air in tube 5,6,7 and 8.

Figure 4. Gas formation in tube 5,6,7 and 8. 30 minutes after adding Sodium pyruvate.

Figure 5. Starting amounts of air in tube 9 and 10.

Figure 6. Gas formation in tube 9 and 10. 30 minutes after adding
Sodium pyruvate.

Figure 7: Gas formation in tube 1, 2, 3 and 4 after a dayr

Figure 8: Gas formation in tube 5, 6, 7 and 8 after a day

Figure 9: Gas formation in tube 9 and 10 after a day

**02**

### Testing gas production

Show more

## August 22

[ ]

# Testing gas production

### August 22

×

Elise Grootscholten | Paul Reusink

Gasproduction testing for the biobricks (K173003 + I13453), (K133071 + I13453) and negative controls (B0015 and K133071 without urea and arabinose).
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and K133071 + I13453 is tested with urea and arabinose for gasproduction.
The negative control also produces a little bit gas.

**Experiment 3: Gasproduction testing**

Gasproduction testing for the biobricks K173003 + I13453, K133071 + I13453 and negative controls.
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and K133071 + I13453 is tested with urea and arabinose for gasproduction.

**Materials**
See experiment 1 and 2.

**Methods**
See experiment 1 and 2.
375 mL culture medium with CAM (already in it) + 18,75 g urea + 0,5 ul arabinose.
340 mL culture medium with CAM (already in it) + 51 mL natriumpyruvate (20 mM) + 0,5 ul arabinose.

**Results and conclusion**
Colonies 5 and 7 of the biobricks K173003 + I13453 (pyruvate)  and number 4 of the biobricks K133071 + I13453 (urea).
In both the negative controls there's been gas produced. Futher gasproduction test will be done.

Figure 1: Starting amounts in tube 1, 2, 3, 4 and 5 with Pyruvate and arabinose

Figure 2: Gasproduction in tube 1, 2, 3, 4 and 5 after a day with pyruvaat and arabinose

Figure 3: Starting amounts in tube 6, 7, 8, 9 and 10 with Pyruvate and arabinose

Figure 4: Gasproduction in tube 6, 7, 8, 9 and 10 after a day with pyruvate and arabinose

Figure 5: Starting amounts in tube 1, 2, 3, 4 and 5 with Urea and arabinose

Figure 6: Starting amounts in tube 6, 7, 8, 9 and 10 with Urea and arabinose

Figure 7: Gasproduction in tube 1, 2, 3, 4 and 5 after a day with urea and arabinose

Figure 8: Gasproduction in tube 6, 7, 8, 9 and 10 after a day with urea and arabinose

Figure 9: Starting amounts in the negative controls B0015 and K133071 + I13453 (without urea and arabinose)

Figure 10: Gasproduction in the negative controls B0015 and K133071 + I13453 after a day (without urea and arabinose)

**03**

### Testing gas production

Show more

## August 22

[ ]

# Testing gas production

### August 24

×

Elise Grootscholten | Paul Reusink

Testing the gasproduction of the colonies 9 and 10 of biobricks (K173003 + J23100), colonies 5 and 7 of biobricks (K173003 + I13453) and colony 4 of biobricks (K133071 + I13453) with and without centrifuging the bacteria. There is also a negative control (J04450 pSB1K3) with Kanamycine. The negative contol started with a lot of gas inside the tube. We can not see wether there is produced more after a day or not. This have to be tested later.

**Experiment 4: Gasproduction testing**
24-08-18

Testing the gasproduction with and without centrifuging the bacteria.

**methods**
See methods experiment 1
2 negative controls
colonies 9 and 10 of biobricks K173003 + J23100.
Colonies 5 and 7 of biobricks K173003 + I13453
Colony 4 of biobricks K133071 + I13453

Culture medium:
150 mL: Sodiumpyruvate (20 mM) 22,5 mL + Chloramphenicol (40 mg/mL) 131 ul
150 mL: Sodiumpyruvate (20mM) 22,5 mL +  Chloramphenicol (40 mg/mL) 131 ul + Arabinose (300 mg/mL) 0,5 ul
80 mL: Urea 4 g + Chloramphenicol (40 mg/mL) 70 ul + Arabinose (300 mg/mL) 0,5 ul
80 mL: Kanamycine (40 mg/mL) 70 ul

Figure 1: Starting amounts Nr. 9, 10 and 4 centrifuged

Figure 2. Gasproduction Nr 9. 10 and 4 after 4 hours centrifuged.

Figure 3: Starting amounts Nr. 5, 7 and negative control centrifuged

Figure 4. Gasproduction Nr 5,7 and negative control after 4 hours centrifuged.

Figure 5: Starting amounts Nr. 9, 10 and 4 not centrifuged

Figure 6. Gasproduction Nr 9. 10 and 4 after 4 hours not centrifuged.

Figure 7: Starting amounts Nr. 5, 7 and negative control not centrifuged

Figure 8. Gasproduction Nr. 5, 7 and negative control after 4 hours, not centrifuged.

**04**

### Testing gas production

Show more

## August 24

[ ]

# Urea and sodium pyruvate test for resistance E.coli

### August 28

×

Randall de Waard | Elise Grootscholten

Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.

**Experiment 2:****Testing different concentrations of Urea and Sodium pyruvate**
**13-08-18**

Same testing like experiment 1 but with different concentrations.

|  |  |  |
| --- | --- | --- |
| **What** | **How much** | **Solution** |
| Urea | 1,2 g | 1 mL purified water |
| Urea | 1,6 g | 2 mL purified water |
| Sodium pyruvate (10 mM) | 10 mL | None |

**Results**

|  |  |  |  |
| --- | --- | --- | --- |
| **What** | **How much** | **Solution** | **Result** |
| Urea | 1,2 g | 1 mL purified water | Little grow |
| Urea | 1,6 g | 2 mL purified water | No grow |
| Sodium pyruvate (10 mM) | 10 mL | - | Grow |
| Positive control | - | - | Grow |
| Negative control | - | - | No Grow |

*Table 2: Results experiment 2*

**Conclusion**

For urea is 1 g used/20 mL and for sodiumpyruvate is 6 mL (10mM) or 3 mL (20 mM) used.

**02**

### Urea and sodium pyruvate test for resistance E.coli

Show more

## August 28

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 30

×

Elise Grootscholten | Randall de Waard

A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.

**Experiment 9: Minipreps**
28-08-18

**Materials**
- Mini prep, Plasmid Purification Kit, Machery Nagel
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

**Methods**
**Miniprep protocol Plasmid Purification Kit, Machery Nagel**
See experiment 3
Make minipreps of the biobricks K352002, K352003

**Results**
The minipreps can not be used, because washing buffer without ethanol is used. The miniprep of K352002 has been done again.

29-08-18

**Methods**
Make a miniprep of the biobrick K352002

**Results**
K352002: 69,77 ng/μL

30-08-18

**Methods**
See experiment 3
Make minipreps of the biobricks K133116 and K173013

Figure 1: Miniprep K352002

**09**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 30

[ ]

# Chemo competent cells

### August 30

×

None

Competent cells of NEB10 Beta and BL21 DE3 were made. These cells were made competent so new biobricks and ligation mixes could be transformed into these cells. BL21 DE3 and NEB10 beta had 80 cups of 50 ul each. 1 cup is needed for 1 transformation later. The cells were competent enough to use in the project.

Making chemocompetent cells:

Materials:
- E. coli NEB10Beta strain
- E. coli Bl21 (DE 3) strain
- Buffer 1

30 mM natriumacetaat, 100mM rubidium chloride,10 mM calciumchloride, 50mM mangaan chloride, 15 % glycerol; pH 5,8 met verdunde azijnzuur, filter steriliseren.

Bewaren bij kamertemperatuur.

- Buffer 2

10 mM MOPS, 75 mM calcium chloride, 10 mM rubidium chloride, 15% glycerol; pH 6,6 met NaOH, filter steriliseren.

Bewaren bij kamertemperatuur

Method:
The day before making competent cells the strains were ented into 20ml LB medium (100ml erlenmeyer) and incubated for 6 hours by 37 degrees at 200 rpm. The culture was then transmitted to 20 ml LB medium (100 ml erlenmeyer) in a dilution of 1:50 (400 ul/ 20 ML) this was incubated overnight at 37 degrees Celsius at 200 rpm.

In the morning the culture was diluted 1:100 in 200 ml LB medium (1L erlenmeyer). This was then incubated by 37 degrees and 225 rpm till a OD600 level of 0,5.
To make sure the right OD was used, the culture was measured every 20/30 minutes. The results of the measurements can be found in part 2.

After hitting the OD600 of 0,5. The cultures were transmitted to 250 ml centrifuge tubes and cooled on ice. The cells are supposed to stay on ice as much as possible after this. After cooling down to 4 degrees, the cultures were centrifuged at 4500 rpm for 10 min at 4 degrees. The supernatant were disposed, the pellet was resuspended in 66,4 ml ice cold Buffer 1(see materials). This was centrifuged  at 5000 rpm for 10 min at 4 degrees.

BL21DE3  80 cups on place: Drawer 3, tower 2, drawer 3, place 2.
NEB10Beta 79 cups on place: Drawer 3, tower 2, drawer 3, place 3.

**01**

### Chemo competent cells

Show more

## August 30

[ ]

# Assessing Gas Production BioBricks in E.Coli

### August 31

×

Elise Grootscholten | Randall de Waard

Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promoters (J23100, I13453, K352002, K352003)  with 4 different gasproduction biobricks (k173003, K173013, K133071, K133116). J23100 is about 1 kb to long. The rest seems likely to be right digested. The ligations will be transformed in NEB10bèta and digested again as control.

**Experiment 10: Digestion, defosforylation and ligation**

**Materials**
-Cutsmart buffer (10x)
-Restriction enzymes XbaI, PstI-HF, EcoRI-HF, SpeI
-Nuclease free water
-DNA
-Phosphatase buffer (10x)
-Phosphatase (1 U/μL)
-Delutionbuffer (5x)

-T4 ligationbuffer (10x)
-T4 DNA ligase (1U/μL)

**Methods**

|  |  |
| --- | --- |
| DNA (J23100 325,59 ng/μl) ~ 2 μg | 6,1 |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Restriction enzyme 2 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 1: Digestion scheme 1*

|  |  |
| --- | --- |
| DNA (J23100 325,59 ng/μl) ~ 2 μg | 6,1 |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 2: Digestion scheme 2*

|  |  |
| --- | --- |
| DNA (J23100 325,59 ng/μl) ~ 2 μg | 6,1 |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 3: Digestion scheme 3*

|  |  |
| --- | --- |
| DNA (K352003 180,65 ng/μl) ~ 2 μg | 11,1 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 2 μL |
| Restriction enzyme 2 (1 U/μL) | 2 μL |
| Nuclease free water | Fill to 50 μL |

*Table 4: Digestion scheme 4*

|  |  |
| --- | --- |
| DNA (K352002 69,77 ng/μl) ~ 2 μg | 28,7 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 2 μL |
| Restriction enzyme 2 (1 U/μL) | 2 μL |
| Nuclease free water | Fill to 50 μL |

*Table 5: Digestion scheme 5*

|  |  |
| --- | --- |
| DNA (K173003 217,06 ng/μL) ~ 2 μg | 9,2 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 2 μL |
| Restriction enzyme 2 (1 U/μL) | 2 μL |
| Nuclease free water | Fill to 50 μL |

*Table 6: Digestion scheme 6*

|  |  |
| --- | --- |
| DNA (I13453 88,18 ng/μL) ~ 2 μg | 22,7 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Restriction enzyme 2 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 7: Digestion scheme 7*

|  |  |
| --- | --- |
| DNA (K133071 186,79 ng/μL) ~ 2 μg | 10,7 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Restriction enzyme 2 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 8: Digestion scheme 8*

|  |  |
| --- | --- |
| DNA (pSB1K3 163,93 ng/μL) ~ 2 μg | 12,2 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 2 μL |
| Restriction enzyme 2 (1 U/μL) | 2 μL |
| Nuclease free water | Fill to 50 μL |

*Table 9: Digestion scheme 9*

|  |  |
| --- | --- |
| DNA (K173013 345,1 ng/μl) ~ 2 μg | 5,8 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Restriction enzyme 2 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 10: Digestion scheme 10*

|  |  |
| --- | --- |
| DNA (K133116 194,1 ng/μl) ~ 2 μg | 10,3 μL |
| Cutsmart (10x) | 5 μL |
| Restriction enzyme 1 (1 U/μL) | 1 μL |
| Restriction enzyme 2 (1 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 11: Digestion scheme 11*

|  |  |  |
| --- | --- | --- |
| **Biobrick** | **Restriction-enzyme 1** | **Restriction enzyme 2** |
| K173003 | XbaI | PstI-HF |
| K173013 | XbaI | PstI-HF |
| K133071 | XbaI | PstI-HF |
| K133116 | XbaI | PstI-HF |
| J23100 | EcoRI-HF | SpeI-HF |
| I13453 | EcoRI-HF | SpeI-HF |
| K352002 | EcoRI-HF | SpeI-HF |
| K352003 | EcoRI-HF | SpeI-HF |
| pSB1K3 | EcoRI-HF | PstI-HF |

*Table 12: Digestion scheme used enzymes*

**Gelelectrophoresis**

|  |  |  |
| --- | --- | --- |
| **Number** | **What** | **Amount** |
| 1. | Ladder | 5 μL |
| 2. | J23100 EcoRI-HF+ SPeI-HF | 5 μL |
| 3. | J23100 EcoRI-HF | 5 μL |
| 4. | J23100 SpeI-HF | 5 μL |
| 5. | pSB1K3 EcoRI-HF + PstI-HF | 5 μL |
| 6 | K173013 XbaI + PstI-HF | 5 μL |
| 7. | K173003 XbaI + PstI-HF | 5 μL |
| 8. | K133071 XbaI + PstI-HF | 5 μL |
| 9. | K133116 XbaI + PstI-HF | 5 μL |
| 10. | K352002 EcoRI-HF + SpeI-HF | 5 μL |
| 11. | K352003 EcoRI-HF + SpeI-HF | 5 μL |
| 12. | I13453 EcoRI-HF + SpeI-HF | 5 μL |
| 13. | Ladder | 5 μL |
| 14. | Ladder | 10 μL |

*Table 13: Gelelectrophoresis order of digestions*

**dephosphorylation**

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 12,5 μL | Vector DNA (0,5 μg) |
| 2 μL | phosphatase buffer (10x) |
| 4,5 μL | milli Q |
| 1 μL | phosphatase (1 U/μL) |
| 20 μL | Total |

*Table 14: Pipetting scheme dephosphorylation*

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 37,5 μL | Vector DNA (0,5 μg) |
| 6 μL | phosphatase buffer (10x) |
| 13,5 μL | milli Q |
| 3μL | phosphatase (1 U/μL) |
| 60 μL | Total |

*Table 15: Pipetting scheme dephosphorylation*

**Pre-Ligationmix:**

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 7,5 μL | Insert DNA (2 μg/50 μL) 300 ng |
| 7,5 μL | Insert DNA (2 μg/50 μL) 300 ng |
| 4 μL | Backbone (0,5 μg/ 20 μL) 100 ng |
| 5 μL | Delutionbuffer (5x) |
| 1 μL | Milli Q |
| 25 μL | Total |

*Table 16: Pre-ligationmix pipette scheme*

**Ligationmix:**

|  |  |
| --- | --- |
| **Volume** | **Compound** |
| 10 μL | Pre-ligationmix |
| 2 μL | 10x T4 ligationbuffer |
| 1 μL | T4 DNA ligase (1U/μL) |
| 8 μL | Milli Q |
| 20 μL | Total |

*Table 17: Ligationmix pipette scheme*

Ligation started at 15.45 (31-08-18) at room temperature.
end ligation around 11.00 (01-09-18)
Store at -20 degrees Celcius.

**10**

### Assessing Gas Production BioBricks in E.Coli

Show more

## August 31

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 3

×

Elise Grootscholten | Randall de Waard

Transformations of the biobrick combinations in NEB10bèta. The transformations are plated on kanamycine agar plates, because all the ligations were done in pSB1K3 (kanamycine resistence) backbone. Pink and with colonies appeared after incubation by 37 degrees Celcius for about 12h. The white colonies will be used for further experiments.

**Experiment 11: transformations of the biobrick ombinations in NEB10bèta.**
03-09-18

**Materials**
See materials experiment 5.
-Kanamycin (40 mg/mL)
-Ampicillin (50 mg/mL)

**Methods**
See methods experiment 5

Make 19 agar plates with kanamycine (Kana) (17,5 ul/20mL) and 1 agar plate with ampicillin (Amp) (40 ul/mL). Plate all the transformations on agar plates with Kana , because all the ligations are done in the pSB1K3 (Kana resistence) backbone.
Devide one Amp plate and one Kana plate in about 30 parts. Because the biobrick combinations with biobrick K133116 have originally Amp and Kana resistence, the right backbone plasmid (with hopefully the right ligation) is only growing on the agar plate with Kana.

**Results**
There is grown n every transformation plates, so the transformations are done well.
There are pink and white colonies present. Only the white colonies will be used for further experiments.

**11**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 3

[ ]

# Soldering PCB [TC]

### September 4

×

Tom van Dijk | Mike Vrieswijk

Soldered the first temperature controller with the help of a sodlering stencil and reflow oven. All components were soldered onto the first temperature controller.

**01**

### Soldering PCB [TC]

Show more

## September 4

[ ]

# Soldering PCB [TC]

### September 4

×

Tom van Dijk | Mike Vrieswijk

Soldered second temperature controller with the help of a sodlering stencil and reflow oven. All components were soldered onto the first temperature controller.

**02**

### Soldering PCB [TC]

Show more

## September 4

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 6

×

Elise Grootscholten | Randall de Waard

Plasmid DNA was isolated of 20 different colonies. The first time something went wrong. The second time we had good concentrations of isolated plasmid DNA. After this we will digest the DNA to see if the plasmids all have the required biobricks. The tube have a code from now on, see table 1.

|  |  |  |  |
| --- | --- | --- | --- |
| **Number** | **Promotor** | **Gene** | **Backbone** |
| A | K352002 (CooF) | K173013 | pSB1K3 |
| B | K352002 (CooF) | K173003 | pSB1K3 |
| C | K352002 (CooF) | K133071 | pSB1K3 |
| D | K352003 (CooM) | K133071 | pSB1K3 |
| E | K352003 (CooM) | K173003 | pSB1K3 |
| F | K352003 (CooM) | K173013 | pSB1K3 |
| H | I13453 | K173013 | pSB1K3 |
| I | I13453 | K173003 | pSB1K3 |
| J | I13453 | K133071 | pSB1K3 |

*Table 1: list of biobricks of abbreviations*

**Experiment 12: Overnight cultures and minipreps**
05-09-18/06-09-18

**Materials**
See materials experiment 3

**Methods**
See methods experiment 3

|  |  |  |  |
| --- | --- | --- | --- |
| **Number** | **Promotor** | **Gene** | **Backbone** |
| A | K352002 (CooF) | K173013 | pSB1K3 |
| B | K352002 (CooF) | K173003 | pSB1K3 |
| C | K352002 (CooF) | K133071 | pSB1K3 |
| D | K352003 (CooM) | K133071 | pSB1K3 |
| E | K352003 (CooM) | K173003 | pSB1K3 |
| F | K352003 (CooM) | K173013 | pSB1K3 |
| G | B0032 | J23105 | pSB1A3 |
| H | I13453 | K173013 | pSB1K3 |
| I | I13453 | K173003 | pSB1K3 |
| J | I13453 | K133071 | pSB1K3 |

Table 1: list of biobricks of abbreviations

Figure 1: Nanodrop results after DNA isolation. The biobrick combinations can be found in the methods (experiment 12)

**12**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 6

[ ]

# Assessing CooA Production

### September 6

×

Dustin van der Meulen

After transforming the ligation product of J23105+B0032 and K352001+B0015 we had some growth of none red/pink colonies. Overnight cultures where made, and the DNA was prepped later on, checking them on gel.

Today a miniprep of pSB1K3 + J23100 + B0032 was performed. This gave concentrations of 51,3 and 89 ng/μL.

Of this, together with pSB1K3+ K352001 + B0015 and pSB1A3 a digest was performed:

|  |  |  |
| --- | --- | --- |
| Biobricks | Enzyme 1 | Enzyme 2 |
| 1K3+J23+B32 | EcoRI | SpeI |
| 1K3+K35+B15 | XbaI | PstI |
| pSB1A3 | EcoRI | PstI |

After two hours the digest was put on gel, though the rest was left for another two hours.

![](/members/experiments/journal/authloader.ashx?fileID=2028112&inline=true&w=500)

WhatsApp Image 2018-09-07 at 15.28.47.jpeg

CooA experiment 14

**14**

### Assessing CooA Production

Show more

## September 6

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 10

×

Elise Grootscholten | Randall de Waard

The minipreps of experiment 12 are digested with the restriction enzymes: SmaI and ScaI. Only Sca and Sma are both incubated at 37 degrees Celcius. Sma has to be incubated at 25 degrees Celcius. This is done in experiment 15.

**Experiment 12: Digestions of the minipreps**
10-09-18

**Materials**
-SmaI (restriction-enzyme)
-ScaI (restriction-enzyme)
See materials experiment 10

**Methods**
See methods experiment 10

|  |  |
| --- | --- |
| DNA (A1 396.7 ng/μl) ~ 2 μg | 5.0 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF (2 U/μL) | 1 μL |
| Sma1 (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 1: Digestion scheme 1*

|  |  |
| --- | --- |
| DNA (B1 15.65 ng/μl) ~ 2 μg | 127.8 |
| Cutsmart (10x) | 5 μL |
| Sca1-HF (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 2: Digestion scheme 2*

|  |  |
| --- | --- |
| DNA (C1 186.6 ng/μl) ~ 2 μg | 10.7 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 3: Digestion scheme 3*

|  |  |
| --- | --- |
| DNA (D1 79.0    ng/μl) ~ 2 μg | 25.3 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 4: Digestion scheme 4*

|  |  |
| --- | --- |
| DNA (E1 97.7 ng/μl) ~ 2 μg | 20.7 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 5: Digestion scheme 5*

|  |  |
| --- | --- |
| DNA (F1 106.7 ng/μl) ~ 2 μg | 18.7 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 6: Digestion scheme 6*

|  |  |
| --- | --- |
| DNA (G1 328.9 ng/μl) ~ 2 μg | 6.1 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 7: Digestion scheme 7*

|  |  |
| --- | --- |
| DNA (H1 121.0 ng/μl) ~ 2 μg | 16.5 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 8: Digestion scheme 8*

|  |  |
| --- | --- |
| DNA (I1 1129.5 ng/μl) ~ 2 μg | 1.7 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 9: Digestion scheme 9*

|  |  |
| --- | --- |
| DNA (J1 1465.7 ng/μl) ~ 2 μg | 1.4 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 10: Digestion scheme 10*

|  |  |
| --- | --- |
| DNA (A2 310.4 ng/μl) ~ 2 μg | 6.4 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 11: Digestion scheme 11*

|  |  |
| --- | --- |
| DNA (B2 364.9l) ~ 2 μg | 5.5 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 12: Digestion scheme 12*

|  |  |
| --- | --- |
| DNA (C2 228.3 ng/μl) ~ 2 μg | 8.8 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 13: Digestion scheme 13*

|  |  |
| --- | --- |
| DNA (D2 701.7 ng/μl) ~ 2 μg | 2.9 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 14: Digestion scheme 14*

|  |  |
| --- | --- |
| DNA (E2 119.8 ng/μl) ~ 2 μg | 16.7 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 15: Digestion scheme 15*

|  |  |
| --- | --- |
| DNA (F2 493.3 ng/μl) ~ 2 μg | 4.1 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 16: Digestion scheme 16*

|  |  |
| --- | --- |
| DNA (G2 211.7 ng/μl) ~ 2 μg | 9.5 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 17: Digestion scheme 17*

|  |  |
| --- | --- |
| DNA (H2 482.2 ng/μl) ~ 2 μg | 4.2 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 18: Digestion scheme 18*

|  |  |
| --- | --- |
| DNA (I2 1364.7 ng/μl) ~ 2 μg | 1.5 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 19: Digestion scheme 19*

|  |  |
| --- | --- |
| DNA (J2 1665.5 ng/μl) ~ 2 μg1.2 | μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF 1 (2 U/μL) | 1 μL |
| Sma1  (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 20: Digestion scheme 20*

**Results**
The results of the digestions can be found in figure 1 and 2 (Images experiment 13)

Ladder, A1, B1, C1, D1, E1, F1, G1, H1, I1, (J1), Ladder, A2, B2, C2, D2, E2, F2, G2, Ladder
Ladder, H2, I2, J2, Ladder, K3, J1, ladder
Something went wrong with the first (J1), so look at the second one.

**Conclusion/discussion**
Restriction-enzyme Sma was incubated at 37 degrees Celcius (same as Sca). This must be 25 degrees Celcius.
Therefore, Sma was added again and was incubated at 25 degrees Celcius. See results experiment 16.

Figure 1: Digestions with ScaI and SmaI

Figure 2: Digestions with ScaI and SmaI

**13**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 10

[ ]

# Soldering PCB [TC]

### September 11

×

Tom van Dijk | Mike Vrieswijk

Testing first temperaturecontroller. Reworking surton components due to shorts on the PCB. Rework succesfull.

**03**

### Soldering PCB [TC]

Show more

## September 11

[ ]

# Soldering PCB [TC]

### September 11

×

Tom van Dijk | Mike Vrieswijk

Testing second temperaturecontroller. Reworking surton components due to shorts on the PCB. Rework failed.

**04**

### Soldering PCB [TC]

Show more

## September 11

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 13

×

Elise Grootscholten | Randall de Waard

Minipreps of the biobrick combinations: K352002+K133116+pSB1K3 (1) and K352003+K133116+pSB1K3 (2) are done. The DNA concentration is for 1:189,3 ng/μl and for 2: 355,3 ng/μl.

**Experiment 14: Minipreps**
13-09-18

Minipreps of the biobrick combinations: K352002+K133116+pSB1K3 and K352003+K133116+pSB1K3

**Materials**
- Mini prep, Plasmid Purification Kit, Machery Nagel
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

**Methods**
Machery Nagel Plasmid Purification
See experiment 3

**Results**
K352002+K133116+pSB1K3: 189,3 ng/μl
K352003+K133116+pSB1K3: 355,3 ng/μl

Figure 1: Minipreps 13-09-18

**14**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 13

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 13

×

Elise Grootscholten | Randall de Waard

Digestions have been done of the biobrickcombinations K352002 +K133116 + pSB1K3 and K352003 + K133116 + pSB1K3 with restriction-enzymes ScaI and SmaI

**Experiment 14: Digestions**
13-09-18

Digestions of the biobrick combinations: K352002+K133116+pSB1K3 and K352003+K133116+pSB1K3

**Materials**
-SmaI (restriction-enzyme)
-ScaI (restricion-enzyme)
See experiment..

**Methods**
1 μL SmaI restriction-enzyme has been added to the digetions from experiment 13, except for pSB1K3.
After an our at room temperature the digestions were put in -20 degrees Celcius.

|  |  |
| --- | --- |
| DNA (K352002+K133116+pSB1K3 189,3 ng/μl) ~ 2 μg | 32,4 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF (2 U/μL) | 1 μL |
| Sma1 (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 1: Digestion scheme 1*

|  |  |
| --- | --- |
| DNA (K352003+K133116\_pSB1K3 355,3 ng/μl) ~ 2 μg | 5,6 μL |
| Cutsmart (10x) | 5 μL |
| Sca1-HF (2 U/μL) | 1 μL |
| Sma1 (2 U/μL) | 1 μL |
| Nuclease free water | Fill to 50 μL |

*Table 2: Digestion scheme 2*

**15**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 13

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 14

×

Elise Grootscholten | Randall de Waard

All the digestions of experiment 13 have digested again with SmaI for an our at 25 degrees Celcius. Because earlier the digestions have been put immediately bt 37 degrees Celcius. It looks like H1, J1 and D2 include the right biobricks.

**Experiment 16: Gelelectroforesis**
14-09-18

**Materials**
-Loading dye (6x)
-2 Log DNA ladder
-Digested DNA
-1% arabinose gel (1 g/100 mL TAE buffer)

**Methods**
Put 1 gram of arabinose with 100 mL TAE buffer.
Heat it in the microwave until the solution becomes clear.
Add 100 μL gelred when the solution is hand warm.
Pour the solution into the gel holder which you taped before.
Wait until the gel is solid then you can pipette your dna with Loading dye into the wells.

L: 3 μL
DNA: 2 μL loading dye + 5 μL DNA, 5 μL on gel.

**Gel 1**
L, A1, B1, C1, D1, E1, F1, G1, G1 with only smaI, H1, I1, J1, A2, B2, C2, D2, E2, F2, G2, L

**Gel 2**
L, H2, I2, J2, L, K352002 + K133116 + pSB1K3,  K352003 + K133116 + pSB1K3

Figure 1: Gel 1 (14-09-18), digested with ScaI and SmaI.

Figure 2: Gel 2, H2, I2, J2, L, K352002+K133116+pSB1K3, K352003+K133116+pSB1K3

**16**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 14

[ ]

# Testing gas production

### September 17

×

Elise Grootscholten | Paul Reusink

Different strains of E.coli are tested as negative control. Those strains were: NEB10Bèta, BL21 (DE3), BL21, HB101, DH5alpha and JM109. NEB10Bèta produced the most gas. BL21, HB101 and JM101 produced none/almost none gas. Because BL21 isn't a K12 strain we can not use that one. That is why we will test further gasproduction in HB101 and JM109. And maybe those will be our final E.coli strains.

**Experiment 5: Testing different strains of E.coli**

**Materials**
All the following types of bacteria are E.coli:
-glycerolstock BL21
-glycerolstock HB101
-glycerolstock DH5alpha
-glycerolstock JM109
-agarplate with NEB10beta
-agarplat with BL21 (DE3)

**Methods**
See experiment 1
Make overnight cultures of the following E.coli tribes: NEB10Bèta, BL21 (DE3), BL21, HB101, DH5alpha, JM109.
All the bacteria are de deluted in normal LB agar, LB agar with urea and LB agar with pyruvate.

**Results**

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Bacteria | LB | LB | Urea | Urea | Pyruvate | Pyruvate |
| NEB10beta | + | +++ | +++ | +++ | + | ++/+++ |
| BL21 (DE3) | - | ++ | - | - | - | - |
| BL21 | - | - | - | - | - | - |
| DH5a | +++ | +/- | - | - | +/- | + |
| HB101 | - | - | - | - | + | - |
| JM109 | - | - | - | - | - | - |

**05**

### Testing gas production

Show more

## September 17

[ ]

# Designing peristaltic pump [PP]

### September 18

×

Tom van Dijk | Mike Vrieswijk

Made a 3d-model of the parts for the peristaltic pump. Parts include: multiple gears, bearings, and casing.

**01**

### Designing peristaltic pump [PP]

Show more

## September 18

[ ]

# 3d-printing peristaltic pump [PP]

### September 18

×

Tom van Dijk | Mike Vrieswijk

3d-printed all parts of the peristaltic pump model.

**02**

### 3d-printing peristaltic pump [PP]

Show more

## September 18

[ ]

# Animating peristaltic pump [PP]

### September 18

×

Tom van Dijk | Mike Vrieswijk

An animation was made of the first peristaltic pump.

**03**

### Animating peristaltic pump [PP]

Show more

## September 18

[ ]

# Assessing CooA Production

### September 20

×

Dustin van der Meulen

This week we peformed another cycle of cloning, starting with digesting J23105+B0032 with EcoRI and SpeI and K352001+B0015 with XbaI and PstI, checking these on gel (which seemed to be correct) and ligating them together on the pSB1A3 version of BBa\_J04450. This was then grown overnight and prepped the following day.

Today a dephos and ligation has been performd using the rAPId dephos and ligation kit and protocol.
For the dehpos a total of 17 μL of pSB1A3 wasused, for an end concentration of 17 ng/μL
For the ligationthe endvolume was trippled as to have enough DNA.
For both inserts (K352001 & B0015 | J23100 + B0032) 7.5 μL was used (150 ng of DNA foreach insert)
For the veccor (pSB1A3) 3 μL was used (51 ng of DNA)
The ligation will be left overnigh (started at 15:30).

**15**

### Assessing CooA Production

Show more

## September 20

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 24

×

Elise Grootscholten | Randall de Waard

32 minipreps have been performed. A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.**Experiment 17: Minipreps**
20-09-18
Overnight cultures (35)
21-09-18
Minipreps (32)

**Materials**
See experiment 3
-Kanamycin (40 mg/mL)

**Methods**
See experiment 3
Make 32 minipreps:
A: 6
B: 6
C:6
D:1
E:6
F:6
H:1
I: 1
J:2

**Results/conclusion**
There was no grow in B6, C2 and C5.
A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.
Thise are: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5

Figure 1: Minipreps, a total of (32) because number 5, 7 and 12 are wrong.

**17**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 24

[ ]

# Integration of silicon hose and peristaltic pump [PP]

### September 25

×

Tom van Dijk | Mike Vrieswijk

Due to ordering issues a different silicon hose was implemented in the design. The new silicon tube was integrated into the 3d-printed peristaltic pump. A test was done with the new silicon tube in place. The test cocluded that the tube was not compatible with this peristaltic pump design. A new design must be made.

**01**

### Integration of silicon hose and peristaltic pump [PP]

Show more

## September 25

[ ]

# Designing peristaltic pump v2 [PP]

### September 25

×

Tom van Dijk | Mike Vrieswijk

Peristaltic pump was redesigned and the model has been updated.

**02**

### Designing peristaltic pump v2 [PP]

Show more

## September 25

[ ]

# 3d-printing peristaltic pump v2 [PP]

### September 25

×

Tom van Dijk | Mike Vrieswijk

3d-printed all parts of the peristaltic pump model v2. All parts have been put together succefully.

**03**

### 3d-printing peristaltic pump v2 [PP]

Show more

## September 25

[ ]

# Testing peristaltic pump v2 [PP]

### September 25

×

Tom van Dijk | Mike Vrieswijk

The silicon tube was intergrated with the 3e-printed peristaltic pump v2 and tested. The test concluded that the motor had insufficient force. A new design must be made.

**04**

### Testing peristaltic pump v2 [PP]

Show more

## September 25

[ ]

# Designing peristaltic pump v3 [PP]

### September 26

×

Tom van Dijk | Mike Vrieswijk

An editional planetary gearset was added to the first planetary gearset of the peristaltic pump v2 model to increase the power output by 4x.

**01**

### Designing peristaltic pump v3 [PP]

Show more

## September 26

[ ]

# 3d-printing peristaltic pump v3 [PP]

### September 26

×

Tom van Dijk | Mike Vrieswijk

3d-printed all parts of the peristaltic pump model v3. All parts have been put together succefully.

**02**

### 3d-printing peristaltic pump v3 [PP]

Show more

## September 26

[ ]

# Testing peristaltic pump v3 [PP]

### September 26

×

Tom van Dijk | Mike Vrieswijk

The newly pot together peristalticpump model v3 was tested. The test concluded a flaw in the design of the peristaltic pump v3. Failure: the silicon tube was pulled through the pump. A new design must be made.

**03**

### Testing peristaltic pump v3 [PP]

Show more

## September 26

[ ]

# Assessing CooA Production

### September 26

×

Dustin van der Meulen

Digesting the CooA combinations with NotI we could check if the requiered biobricks where present in our cells. Out of 10 checked colonies, 3 appeared to be the right size. Because of an error the on plate location of these colonies was lost however, so another check will have to be performed.

Pipet 8 digestions
Nr. 1, 2, 4, 5, 7, 8, 9, and 10 of CooA. Use 5 μL of digest with 3 μL of loading dye.

Gel:
Ladder 5 ul 1, 2, 4, 5, 7, 8, 9, 10, pSB1K3 (Sca), G1 (Sma), Ladder 5 ul, ladder 3 ul
The CooA digestions are 5 ul

![](/members/experiments/journal/authloader.ashx?fileID=2101903&inline=true&w=500)

Gel CooA.jpg

CooA 18-09

**16**

### Assessing CooA Production

Show more

## September 26

[ ]

# Designing Test tube shaker [TTS]

### September 27

×

Tom van Dijk | Mike Vrieswijk

A test tube shaker was designed to fit on top of a NEMU17 stepper motor. A spot for end switch was put in place to calibrate the test tube shaker. A cap for the test tube was designed.

**01**

### Designing Test tube shaker [TTS]

Show more

## September 27

[ ]

# 3d-printing Test tube shaker [TTS]

### September 27

×

Tom van Dijk | Mike Vrieswijk

The test tube shaker was 3d-printed and assembled. Nine caps were printed.

**02**

### 3d-printing Test tube shaker [TTS]

Show more

## September 27

[ ]

# Testing Test tube shaker [TTS]

### September 27

×

Tom van Dijk | Mike Vrieswijk

The newly assembled testtube shaker was tested for sufficient and controlled shakage. The test was deemed a succes. The calibration for the test tube shaker potition was tested and deemed in working order. The caps were tested for sufficient air permeability. The test concluded suficient air permeability.

**03**

### Testing Test tube shaker [TTS]

Show more

## September 27

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 27

×

Elise Grootscholten | Randall de Waard

Making overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin. These biobrick combinations can be found in experiment 12. Also the first digestions with XbaI, Eco0109I, HindIII and SspI-HF have been done.

**Experiment 19: Making overnight cultures + first digestions**
27-09-18

**Materials**
-Kanamycine
-XbaI
-SspI-HF
-Eco019I
-HindIII

**Methods**
Make new overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin.

**19**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 27

[ ]

# Assessing Gas Production BioBricks in E.Coli

### September 27

×

Elise Grootscholten | Randall de Waard

Minipreps have been made of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.

**Experiment 20: Minipreps of gasproduction biobricks**
27-09-18

Make minipreps of A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.
See experiment 3.

Also make master mixes for the control digestions and code the tubes.

**20**

### Assessing Gas Production BioBricks in E.Coli

Show more

## September 27

[ ]

# Designing peristaltic pump v4 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

The innershape of the tube duct was rounded to increase friction on the tube. The model of the peristaltic pump v3 was updated.

**01**

### Designing peristaltic pump v4 [PP]

Show more

## September 28

[ ]

# 3d-printing peristaltic pump v4 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

The new parts for the tubeduct were 3d-printed and put together with the peritstaltic pump.

**02**

### 3d-printing peristaltic pump v4 [PP]

Show more

## September 28

[ ]

# Testing peristaltic pump v4 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

The new peristaltic pump v4 was tested. The test concluded a large improvement over the previous generation, but was deemed unreliable due tothe tube occasional being pulled through the pump. A new design must be made.

**03**

### Testing peristaltic pump v4 [PP]

Show more

## September 28

[ ]

# Designing peristaltic pump v4.1 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

A clamp was designed to hold the tube into the peristaltic pump from both ends. the peristaltic pump model v4 was updated.

**04**

### Designing peristaltic pump v4.1 [PP]

Show more

## September 28

[ ]

# 3d-printing peristaltic pump v4.1 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

The new clamp was 3d-printed, put together and added to the epristaltic pump.

**05**

### 3d-printing peristaltic pump v4.1 [PP]

Show more

## September 28

[ ]

# Testing peristaltic pump v4.1 [PP]

### September 28

×

Tom van Dijk | Mike Vrieswijk

The new peristaltic pump v4.1 was tested. The test conclude no noticable difference to the behaviour of the tube. The tube was still occsionally pulled through the pump. A new design must be made.

**06**

### Testing peristaltic pump v4.1 [PP]

Show more

## September 28

[ ]

# Designing peristaltic pump v5 [PP]

### September 30

×

Tom van Dijk | Mike Vrieswijk

The tube clamp was attached to the top piece of the model's casing. The model of the persitaltic pump v4.1 was updated.

**01**

### Designing peristaltic pump v5 [PP]

Show more

## September 30

[ ]

# 3d-printing peristaltic pump v5 [PP]

### September 30

×

Tom van Dijk | Mike Vrieswijk

The new integrated tube clamp was 3d-printed and added to the peristaltic pump.

**02**

### 3d-printing peristaltic pump v5 [PP]

Show more

## September 30

[ ]

# Testing peristaltic pump v5 [PP]

### September 30

×

Tom van Dijk | Mike Vrieswijk

The new peristaltic pump v5 was tested. The new v5 was deemed a succes. No previous bad behaviour was observed.

**03**

### Testing peristaltic pump v5 [PP]

Show more

## September 30

[ ]

# Assessing CooA Production

### October 1

×

Dustin van der Meulen

Today a colony PCR was performed of multiple CooA ligation products of J23100+B0032+K352001+B0015 on pSB1A3. This specific colony was later PCRed again with the gasproduction biobricks in preperation for sequencing. Only one of the colonies seemed to be the right length, though te lenght of the complete insert and BBa\_J04450 is quite similair, so sequencing later this week will provide the final answer.

CooA 4-10

**17**

### Assessing CooA Production

Show more

## October 1

[ ]

# Assessing Gas Production BioBricks in E.Coli

### October 4

×

Elise Grootscholten | Randall de Waard

From 27-09 until 04-10 more than 200 digestions and 17 gelelectrophoresis have been performed. This was done as a control for our composite parts. We digested with XbaI, Eco01019 and SspI-HF/HindIII. Every restriction-enzyme has an unique place to cut in our constructs. With this we could test if the promoter, backbone and gas production gene was present. However, the digestions didn't show us very good results. Only I1 and J1 looked good. After this we started with PCR.

**Experiment 21: Digestions and gelelectrophoresis**
01-10-18

Digestions of the gasproduction biobrick combinations (D, H, I and J) and a start with the gelelectrophoresis.

**Methods**
Digestions of D2, H1, I2 and J2 of experiment 12.
J2 will from now on be J3.

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 4 μL |
| Eco0109I (20 U/μL) | 1 μL |
| Nuclease free water | 35 μL |

*Table 1: Master mix digestion 1*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 4 μL |
| Eco0109I (20 U/μL) | 1 μL |
| XbaI (20 U/μL) | 1 μL |
| Nuclease free water | 34 μL |

*Table 2: Master mix digestion 2*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| Eco0109I (20 U/μL) | 1 μL |
| XbaI (20 U/μL) | 1 μL |
| HindIII (20 U/μL) | 1 μL |
| Nuclease free water | 15 μL |

*Table 3: Master mix digestion 3*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| Eco0109I (20 U/μL) | 1 μL |
| XbaI (20 U/μL) | 1 μL |
| SspI-HF | 1 μL |
| Nuclease free water | 15  μL |

*Table 4: Master mix digestion 4*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| HindIII (20 U/μL) | 1 μL |
| XbaI (20 U/μL) | 1 μL |
| Nuclease free water | 16 μL |

*Table 5: Master mix digestion 5*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| SspI-HF (20 U/μL) | 1 μL |
| XbaI (20 U/μL) | 1 μL |
| Nuclease free water | 16 μL |

*Table 6: Master mix digestion 6*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 4 μL |
| XbaI (20 U/μL) | 1 μL |
| Nuclease free water | 35 μL |

*Table 7: Master mix digestion 7*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| HindIII (20 U/μL) | 1 μL |
| Nuclease free water | 17 μL |

*Table 8: Master mix digestion 8*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| SspI-HF (20 U/μL) | 1 μL |
| Nuclease free water | 17 μL |

*Table 9: Master mix digestion 9*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| Eco0109I (20 U/μL) | 1 μL |
| HindIII (20 U/μL) | 1 μL |
| Nuclease free water | 16 μL |

*Table 10: Master mix digestion 10*

|  |  |
| --- | --- |
| NEB 2.1 (10x) | 2 μL |
| Eco0109I (20 U/μL) | 1 μL |
| SspI-HF (20 U/μL) | 1 μL |
| Nuclease free water | 16 μL |

*Table 11: Master mix digestion 11*

Pipette 9 μL of the mastermix with 2 μL of H1/ 1 μL of D2/ 1 μL of I2/ 1 μL of J23

D2 and J3 are digested with mastermix: 1, 2, 3, 5, 7, 8 and 10.
H1 and I2 are digested with mastermix: 1, 2, 4, 6, 7, 9, and 11.

L= 2 log DNA ladder

**Gelelectrophoresis**
01-10-18

**Gel 1**
L, B6 Eco01090I, B6 SspI-HF, B6 XbaI, B6 Eco0109I + SspI-HF, B6 Eco0109I + XbaI, B6 SspI-HF + XbaI, B6 Eco0109I + SspI-HF + XbaI, L, B2 SspI-HF, B2 XbaI, B2 Eco0109I + SspI-HF, B2 Eco0109I + XbaI, B2 SspI-HF + XbaI, B2 Eco0109I + SspI-HF + XbaI, L, H1 SspI-HF, H1 XbaI, H1Eco0109I + SspI-HF, H1 Eco0109I + XbaI

**Gel 2**
L, H1 SspI-HF + XbaI, H1 Eco0109I + SspI-HF + XbaI, I1 Eco0109I + SspI-HF, I1 Eco0109I + XbaI, I1 SspI-HF + XbaI, I1 Eco0109I + SspI-HF + XbaI, L, I1 SspI-HF, I1 XbaI, B3 SspI-HF, B3 XbaI, B3 Eco0109I + SspI-HF, B3 Eco0109I + XbaI, B3 SspI-HF + XbaI, B4 SspI-HF, B4 XbaI, B4 Eco0109I + SspI-HF, B4 Eco0109I + XbaI, B4 Eco0109I + SspI-HF + XbaI

**Gel 3**
L, F6 SspI-HF, F6 XbaI, F6 Eco0109I + SspI-HF, F6 Eco0109I + XbaI, F6 SspI-HF + XbaI, F6 Eco0109I + SspI-HF + XbaI, L, F5 SspI-HF, F5 XbaI, F5 Eco0109I + SspI-HF, F5 Eco0109I + XbaI, F5 SspI-HF + XbaI, F5 Eco0109I + SspI-HF + XbaI, L F4 XbaI, F4 Eco0109I + SspI-HF, F4 Eco0109I + XbaI, F4 sspI-HF + XbaI, L

**Gel 4**
L, F4 Eco0109 + XbaI + SspI-HF, F4 Eco0109, F3 XbaI, F3 Eco0109 + SspI-HF, F3 Eco0109 + XbaI, F3 SspI-HF + XbaI, F3 Eco0109 + XbaI + SspI-HF, F2 SspI-HF, F2 XbaI, L, F2 Eco0109 + SspI-HF, F2 Eco0109 + XbaI, F2 SspI-HF + XbaI, F2 Eco01090 + XbaI + SspI-HF, F1 XbaI, F1 Eco0109 + SspI-HF, F1 Eco0109 + XbaI, F1 SspI-HF + XbaI

**Gel 5**
L, F1 Eco0109 + XbaI + SspI-HF, E1 Eco0109, E1 SspI-HF, E1 XbaI, E1 Eco0109 + SspI-HF, E1 Eco0109 + XbaI, E1 SspI-HF + XbaI, E1 Eco0109 + XbaI + SspI-HF, L, E2 Eco0109, E2 SspI-HF, E1 XbaI, E2 Eco0109 + SspI-HF, E2 Eco0109 + XbaI, E2 SspI-HF + XbaI, E2 Eco0109 + XbaI + SspI-HF, L

**Gel 6**
L, E3 SspI-HF, E3 XbaI, E3 Eco0109 + SspI-HF, E3 Eco0109 + XbaI, E3 SspI-HF + XbaI, E3 Eco0109 + XbaI + SspI-HF, L, E6 Eco0109, E6 SspI-HF, E6 XbaI, E6 Eco0109 + SspI-HF, E6 Eco0109 + XbaI, E6 SspI-HF + XbaI, E6 Eco0109 + XbaI + SspI-HF, L

02-10-18
**Gelelectrophoresis**

**Gel 1**
L,- , A1 Eco0109 , A1 SspI-HF, A1 XbaI, A1 Eco0109 + SspI-HF, A1 Eco01019 + XbaI, A1 SspI-HF + XbaI, A1 Eco0109 + XbaI + SspI-HF, -, -, A2 Eco0109 , A2 SspI-HF, A2 XbaI, A2 Eco0109 + SspI-HF, A2 Eco01019 + XbaI, A2 SspI-HF + XbaI, A2 Eco0109 + XbaI + SspI-HF, -, L

**Gel 2**
L,- , A3 Eco0109 , A3 SspI-HF, A3 XbaI, A3 Eco0109 + SspI-HF, A3 Eco01019 + XbaI, A3 SspI-HF + XbaI, A3 Eco0109 + XbaI + SspI-HF, -, -, A4 Eco0109 , A4 SspI-HF, A4 XbaI, A4 Eco0109 + SspI-HF, A4 Eco01019 + XbaI, A4 SspI-HF + XbaI, A4 Eco0109 + XbaI + SspI-HF, -, L

03-10-18
**Gelelectrophoresis**

**Gel 1**
L, D2 Eco0109, D2 Eco0109 + XbaI, D2 Eco0109 +XbaI + HindIII, D2 HindII + XbaI, D2 XbaI, D2 HindIII, D2 Eco0109 + HindIII, D not digested, L, J3 Eco0109, J3 Eco0109 + XbaI, J3 Eco0109 + XbaI + HindIII, J3 XbaI, J3 HindIII, J3 Eco0109 + HindIII, J3.10, J not digested, L.

**Gel 2**
L, H2 Eco0109, H2 Eco0109 + XbaI, H2 Eco0109 + XbaI + SspI-HF, H2 SspI-HF + XbaI, H2 XbaI, H2 SspI-HF, H2 Eco0109 + SspI-HF, H2 not digested, L, I2 Eco0109, I2 Eco0109 + XbaI, I2 Eco0109 + XbaI + SspI-HF, I2 SspI-HF + XbaI, I2 XbaI, I2 SspI-HF, I2 Eco0109 +SspI-HF, I2 not digested, L

**Gel 3**
L, C1 Eco0109, C1 HindIII, C1 XbaI, C1 Eco0109 + XbaI, C1 Eco0109 + HindIII, C1 HindIII + XbaI, C1 Eco0109 + XbaI + HindIII, C1 not digested, L, C2 Eco0109, C2 HindIII, C2 XbaI, C2 Eco0109 + XbaI, C2 Eco0109 + HindIII, C2 HindIII + XbaI, C2 Eco0109 + XbaI + HindIII, C2 not dgested, L

**Gel 4**
L, C3 Eco0109, C3 HindIII, C3 XbaI, C3 Eco0109 + XbaI, C3 Eco0109 + HindIII, C3 HindIII + XbaI, C3 Eco0109 + XbaI + HindIII, C3 not digested, L, C4 Eco0109, C4 HindIII, C4 XbaI, C4 Eco0109 + XbaI, C4 Eco0109 + HindIII, C4 HindIII + XbaI, C4 Eco0109 + XbaI + HindIII, C4 not dgested, L

**Gel 5**
L, C6 Eco0109, C6 HindIII, C6 XbaI, C6 Eco0109 + XbaI, C6 Eco0109 + HindIII, C6 HindIII + XbaI, C6 Eco0109 + SspI-HF + XbaI, C6 not digested, L, B5 Eco0109, B5 SspI-HF, B5 XbaI, B5 XbaI (other tube), B5 Eco0109 + XbaI, B5 SspI-HF + Eco0109, B5 SspI-HF + XbaI, B5 Eco0109 + SspI-HF + XbaI, L

04-10-18
**Gelelectrophoresis**

**Gel 1**
L, J2 Eco0109, J2 HindIII, J2 XbaI, J2 Eco0109 + XbaI, J2 Eco0109 + HindIII, J2 HindIII + XbaI, J2 Eco0109 + HindIII + XbaI, J2 not digested, L, J1 Eco0109, J1 HindIII, J1 XbaI, J1 Eco0109 + XbaI, J1 Eco0109 + HindIII, J1 HindIII + XbaI, J1 Eco0109 + HindIII + XbaI, J1 not digested, L

**Gel 2**
L, B2 Eco0109, B2 HindIII, B3 Eco0109, B3 HindIII, B3 Eco0109 + SspI-HF + XbaI, B4 Eco0109, B4 HindIII, B4 Eco0109 + SspI-HF + XbaI, not digested B2, L, D1 Eco0109, D1 HindIII, D1 XbaI, D1 Eco0109 + XbaI, D1 Eco0109 + HindIII, D1 HindIII + XbaI, D1 Eco0109 + HindIII + XbaI, not digested D1, L

**Gel 3**
L, F1 Eco0109, F1 HindIII, F2 Eco0109, F3 Eco0109, F3 HindIII, F6 Eco0109, F6 HindIII not digested F6, L, A2 HindIII, A3 HindIII, not digested A1, E3 Eco0109, E3 HindIII, not digested E3, L

**Gel 4**
L, H1 Eco0109, H1 HindIII, H1 not digested, I1 Eco0109, I1 HindIII, L

Figure 1: Gel 1 01-10-18

Figure 2: Gel 2 01-10-18

Figure 3: Gel 3 01-10-18

Figure 4: Gel 4 01-10-18

iFigure 5: Gel 5 01-10-18

iFigure 6: Gel 6 01-10-18

Figure 7: Gel 1 02-10-18

Figure 8: Gel 2 02-10-18

Figure 9: Gel 1 03-10-18

Figure 10: Gel 2 03-10-18

Figure 11: Gel 3 03-10-18

Figure 12: Gel 4 03-10-18

Figure 13: Gel 5 03-10-18

Figure 14: Gel 1 04-10-18

Figure 15: Gel 2 04-10-18

Figure 16: Gel 3 04-10-18

Figure 17: Gel 4 04-10-18

**21**

### Assessing Gas Production BioBricks in E.Coli

Show more

## October 4

[ ]

# Preparing DNA for Submission

### October 5

×

Dustin Vermeulen | Elise Grootscholten | Randall de Waard

Today we made colony PCRs of the biobricks we suspected that where correct of our gasproduction, and of colony 8 of CooA. These where then send to the Erasmus Medical Centre, who sequenced our parts for us.

**Colony PCR**
Materials: For our PCR (Both plasmid and colony) reactions we have used the standard neb protocols for a 25 μl reaction , with the following concentrations:

10X Standard Taq Reaction Buffer        2.5 μl
10 mM dNTPs                                        0.5 μl
10 μM forward Primer  (VF2)                 0.5 μl
10 μM reverse Primers  (VR)                 0.5 μl
Taq DNA Polymerase                             0.125 ul
Nuclease-free water                               to 25 μl

The primers VF2 and VR got delivered in powder form. So it was dissolved in Nuclease-free water till a concentration of 100 μm.

Methods: A master mix is made with the concentrations shown above. In case of a plasmid pcr, a volume of plasmid is added before the reaction is filled with Nuclease-free water. With the colony PCR we used a toothpick to touch on the desired colony. Before briefly submerging and shaking the tip of the toothpick in the PCR Eppendorfcup filled with the mastermix. Before pricking it into a clean Agar plate a couple of times to grow more colonies in case the PCR shows us the desired products

For our PCR reactions we use the Bio-Rad T100 thermal cycler. Afterwards the products of the PCR reaction were ran on a gel electrophoresis.

**PCR Purification**
8 μL PCR product + 2 μL ExoSAP-IT
37°C 20 minutes
80°C 15 minutes

**Sequencing Reaction**

|  |  |
| --- | --- |
| 4,0 μL | PCR product |
| 0,15 μL | forward or reverse primer |
| 0,5 μL | BigDye V3.1 (Applied Biosystems) |
| 1,75 μL | Sequencing buffer (5x) |
| 3,6 μL | Nuclease free water |
| 10 μL | Total |

96°C 1 minute
96°C 10 seconds
55°C 5 seconds
60°C 2 minutes
run for 35 cycli
10°C end

**Sequencing reaction purification**
Following protocol of CleanSeq Agencourt

**Sequencing**
ABI 3730 XL, 50 cm capillair

**01**

### Preparing DNA for Submission

Show more

## October 5

[ ]

# Preparing DNA for Submission

### October 7

×

Dustin Vermeulen | Elise Grootscholten | Randall de Waard

Today we digested our succesfully sequenced biobricks (B1 / B6 (same biobrick), D1, I1 and J2), using EcoRI and PstI to get them into a pSB1C3 backbone. This was then transormed and plated. And the end of the day we made 6 overnight cultures of the barely visible colonies.

**Materials**
-Restriction enzymes EcoRI-HF and PstI

**Methods**
Digestion

Mastermix

|  |  |
| --- | --- |
| Cutsmart (10x) | 55 μL |
| EcoRI-HF (20 U/μL) | 1 μL |
| PstI (20 U/μL) | 1 μL |
| Nuclease free water | 382,7 μL |

50 μL mastermix - ... μL DNA (see below)
A2: 12,6 μL
B1: 15,7 μL
B5: 9,4 μL
B6: 9,6 μL
D1: 10,2 μL
E1: 15,7 μL (1 μg)
I1: 14,3 μL (1 μg)
J1.2: 17,4 μL
CooA (8): 5,4 μL

Dephosphorylation + ligation using rAPId dephos and ligation protocol and kit

**02**

### Preparing DNA for Submission

Show more

## October 7

[ ]

# Preparing DNA for Submission

### October 9

×

Dustin Vermeulen | Elise Grootscholten | Randall de Waard

Today we mini prepped our DNA (~6 colonies per succesfully sequenced biobrick, overnight cultures made on the 8th of october) and digested this.

**03**

### Preparing DNA for Submission

Show more

## October 9

[ ]

# Testing gas production

### October 11

×

Elise Grootscholten | Paul Reusink

Overnight cultures made of the following strains with the right plasmid:

|  |  |  |  |
| --- | --- | --- | --- |
| **Number** | **BBa\_** | **HB101** | **JM109** |
| J1 | BBa\_K2604000 | 1x | 1x |
| I1 | BBa\_K2604001 | 3x | 3x |
| D1 | BBa\_K2604003 | 2x | 2x |
| B1 | BBa\_K2604006 | 1x | 1x |
| B6 | BBa\_K2604006 | 1x | 1x |

*Table 1: Overnight culture scheme.*

I1, D1 and J1 are tested for the gas productio. The rest is only made for glycerolstocks

We can not say for sure if the PBad promoter and the gas production enzyme pyruvate decarboxylase work. Fot that more gas production test have to be done. Also the negative controls produce gas. This is a problem. The JM109 and HB101 E.coli strains didn't produce gas in experiment .

**Experiment 6: Testing gasproduction with our new biobricks.**

Testing our new biobricks K260401 in JM109 and HB101. As a negative control we used the other new biobricks ...

**Materials**
-LB culture medium
-Kanamycin
-Bacteria on agar plates with the right plasmid

**Methods**
Make overnight cultures of the following strains with the right plasmid:

|  |  |  |  |
| --- | --- | --- | --- |
| **Number** | **BBa\_** | **HB101** | **JM109** |
| J1 | BBa\_K2604000 | 1x | 1x |
| I1 | BBa\_K2604001 | 3x | 3x |
| D1 | BBa\_K2604003 | 2x | 2x |
| B1 | BBa\_K2604006 | 1x | 1x |
| B6 | BBa\_K2604006 | 1x | 1x |

*Table 1: Overnight culture scheme.*

Only the numbers J1, I1 and D1 are tested. The rest is made for making glycerolstocks.
J1 and D1 are the negative controls in this experiment.

LB: 582 mL +18 mL 100 mM sodiumpyruvate and 525 μL kanamycin (40 mg/mL).
After this the LB is devided in 2. Number 1: 0,5 μL arabinose, number 2: 1,0 μL arabinose.
The rest is done just like earlier experiments with a total of 20 tubes because everthing is done twice.

**Results**
Some of the I1 (BBa\_K2604001) JM109 and HB101 bacteria and the negative controls D1 and J1 (BBa\_K2604003 and BBa\_K2604000) have produced gas over the weekend.
We can not say for sure if the PBad promoter and the gas production enzyme pyruvate decarboxylase work. Fot that more gas production test have to be done. Also the negative controls produce gas. This is a problem. The JM109 and HB101 E.coli strains didn't produce gas in experiment .

Figure 1: Gas testing before the weekend, II1.1 HB101 nr.1/ I1.2 HB101 nr.1/ I1.3, HB101 nr.1/ I1.1 JM109 nr.1

Figure 2: Gas testing after the weekend, II1.1 HB101 nr.1/ I1.2 HB101 nr.1/ I1.3, HB101 nr.1/ I1.1 JM109 nr.1

Figure 3: Gas testing before the weekend, I1.3 JM109 nr.1/ I1.2 JM109 nr.1/ J1 JM109 nr.1/ D1 HB101 nr.1

Figure 4: Gas testing after the weekend, I1.3 JM109 nr.1/ I1.2 JM109 nr.1/ J1 JM109 nr.1/ D1 HB101 nr.1

Figure 5: Gas testing before the weekend, D1 JM109 nr.1/ J1 HB101 nr.1

Figure 6: Gas testing after the weekend, D1 JM109 nr.1/ J1 HB101 nr.1

Figure 7: Gas production before the weekend, I1.3 JM109 nr.2/ I1.2 JM109 nr.2/ I1.1 JM109 nr.2/ I1.3 HB101 nr.2

Figure 8: Gas production after the weekend, I1.3 JM109 nr.2/ I1.2 JM109 nr.2/ I1.1 JM109 nr.2/ I1.3 HB101 nr.2

Figure 9: Gas production before the weekend, D1 JM109 nr.2/ D1 HB101 nr.2/ J1 HB101 nr.2/ J1 JM109 nr.2

Figure 10: Gas production after the weekend, D1 JM109 nr.2/ D1 HB101 nr.2/ J1 HB101 nr.2/ J1 JM109 nr.2

Figure 11: Gas production before the weekend, I1.2 HB101 nr.2/ I1.1 HB101 nr.2

Figure 12: Gas production after the weekend, I1.2 HB101 nr.2/ I1.1 HB101 nr.2

**06**

### Testing gas production

Show more

## October 11

[ ]

# Assessing different ATP sensors

### October 15

×

Loraine Nelson | Elise Grootscholten | Paul Reusink

Biobricks from iGEM were transformed into NEB10Beta. A few of these are sensors that react to ATP and can be used to detect living cells. The Bacteria were plated on agar plates with the right antibiotics to select the bacteria with the right plasmid in them. Every transformation was successful, since there were over 300 colonies on every plate. These colonies could than be used for further experiments and can be easily used for DNA isolation.

Materials:

- Biobricks
- Nuclease free water
- LB- agar
- Variable Volume Pipettes
- Sterile pipetpoints
- Chemically competent cells ( NEB10Beta, 1x10^9)
- Heater (42°C)
- Incubator + shaker (37°C and 250 rpm)
- Eppendorf tubes (1,5 and 2 ml)
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium

Method:

To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24°C). After incubation the DNA is transferred to Eppendorf tubes and put on ice.

For the transformation 1 ul of the DNA was added to 50 ul chemically competent cells strain NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube in a heater (42°C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes.
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37°C and 250 rpm.

After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37°C.

Results:
Tabel 1 shows the results of the plates which were incubated.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Biobricks: | Antibiotics: | Dilution: | Colonies: | CFU: |
| Bba-K352002 | Can | 0x | >300 | - |
| Bba-K352002 | Can | Centrifuged | >300 | - |
| Bba-K352003 | Can | 0x | >300 | - |
| Bba-K352003 | Can | Centrifuged | >300 | - |
| Bba-K352004 | Can | 0x | >300 | - |
| Bba-K352004 | Can | Centrifuged | >300 | - |
| Bba-K352005 | Can | 0x | >300 | - |
| Bba-K352005 | Can | Centrifuged | >300 | - |
| Bba-K352006 | Can | 0x | >300 | - |
| Bba-K352006 | Can | Centrifuged | >300 | - |
| Bba-K352007 | Can | 0x | >300 | - |
| Bba-K352007 | Can | Centrifuged | >300 | - |
| Bba-K352008 | Can | 0x | >300 | - |
| Bba-K352008 | Can | Centrifuged | >300 | - |
| Bba-K352009 | Can | 0x | >300 | - |
| Bba-K352009 | Can | Centrifuged | >300 | - |
| Bba-K1390001 | Can | 0x | >300 | - |
| Bba-K1390001 | Can | Centrifuged | >300 | - |
| Bba-K1390003 | Can | 0x | >300 | - |
| Bba-K1390003 | Can | Centrifuged | >300 | - |
| Bba-K1023003 | Can | 0x | >300 | - |
| Bba-K1023003 | Can | Centrifuged | >300 | - |
| Bba-K284001 | amp | 0x | >300 | - |
| Bba-K284001 | amp | Centrifuged | >300 | - |
| Positive controle: Bba- K1023003 | None | 0x | >300 | - |
| Negative controle: outgrow medium | none | 0x | 0 | - |

Tabel 1: results from the transformation with the biobricks.

Showed in the Tabel, the transformation gave  enough colonies on each plate, which can be used for further experiments. There is no contamination, as seen by the negative control.

Conclusion:

The transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4°C and can be used for further experiments.

**04**

### Assessing different ATP sensors

Show more

## October 15

[ ]

# Assessing different ATP sensors

### October 15

×

Loraine Nelson | Elise Grootscholten | Paul Reusink

The bacteria were mini prepped and than transformed into BL21 DE3. BL21 DE3 has a T7 promotor which is needed for expression of the biobricks. The transformation was successful and multiple colonies were formed on the agar plates with antibiotics. From this plates we grow a few colonies onto new plates for further experiments.

# Transformation Protocol NEB Overview

Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs.

## Protocol

1. Thaw competent cells on ice.
2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3. Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4 to 5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds\*. Do not mix.
6. Add 950 μl of room temperature media\* to the tube.
7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Spread 50 to100 μl of the cells and ligation mixture onto the plates.
10. Incubate overnight at 37°C.

    \* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells� manufacturer.

    1 ul isolated K1023003 biobrick + 50 ul NEB10bèta
    Positive and negative control without antibiotics

**05**

### Assessing different ATP sensors

Show more

## October 15

[ ]

# Assessing Gas Production BioBricks in E.Coli

### October 16

×

Elise Grootscholten | Randall de Waard

Glycerolstocks have been made of several bacteria with plasmids we created or from the iGEM kit.

**22**

### Assessing Gas Production BioBricks in E.Coli

Show more

## October 16

[ ]

# Assessing CooA Production

### October 16

×

Dustin van der Meulen

After performing a digestion on the 16th we ran a gel electrophoresis, from which we could conlcude that only the combinations of J23100+B0031, J23100+B0032 and K352001+B0015 seemed to be correct, though because of the small insert size in the promoter and rbs combinations it is difficult to say. From these combinations overnight cultures where made, which we prepped today.

|  |  |
| --- | --- |
| J23100+B0031 | 125,9 ng/uL |
| J23100+B0032 | 334,1 ng/μL |
| K352001+B0015 | 240,3 ng/μL |

After this we've discussed our progress with our PI's,

**08**

### Assessing CooA Production

Show more

## October 16

[ ]

# Chemo competent cells

### October 16

×

None

New competent cells were made. This time we used the strains JM109 and HB101, since these strains don't produce gas of themselfes. These strains can be used for measurement on our gas production system. The cells are competent enough to use.

**Experiment 2: Chemocompetent cells of E.coli strains JM109 and HB101**
28-09-18

**Methods**
The methods are for JM109 and HB101 both apart.
2 ul pUC19 + 50 ul competent cells
With ampicilin: not diluted, 10^-1, 10^-2, 10^-3, 10^-4
Without ampicillin: not diluted, 10^-4, 10^-5, 10^-6
Negative control: SOC medium.

200 ul not diluted -->
30 ul + 270 ul SOC (10^-1) -->
30 ul + 270 ul SOC (10^-2) -->
30 ul + 270 ul SOC (10^-3) -->
30 ul + 270 ul SOC (10^-4) -->
30 ul + 270 ul SOC (10^-5) -->
30 ul + 270 ul SOC (10^-6)

Results
JM109: 3,4\*10^6 cfu/ug DNA
HB101: 4,4\*10^6 cfu/ug DNA

**02**

### Chemo competent cells

Show more

## October 16

[ ]

# Preparing DNA for Submission

### October 17

×

Dustin Vermeulen | Elise Grootscholten | Randall de Waard

Today we set our control digest from the 9th of october on gel (the gel was inconclusive because of poor quality), after which we put ~500 ng of DNA on our 96 wells submission plate (both the pSB1C3 and pSB1K3 (sequenced) versions of our biobricks). This was then dried in a 50°C stove for around 2 hours (until all wells were drie).

**04**

### Preparing DNA for Submission

Show more

## October 17

# Relevant pages

[## Results](https://2018.igem.org/Team%3ARotterdam_HR/Results)
[## Hardware](https://2018.igem.org/Team%3ARotterdam_HR/Hardware)
[## Software](https://2018.igem.org/Team%3ARotterdam_HR/Software)
[## Experiment](https://2018.igem.org/Team%3ARotterdam_HR/Experiments)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Rotterdam HR/Experiments

# Experiments

# Protocols

Protocol 1 : NucleoSpin® Plasmid EasyPure protocol mini (Marchery Nagel)

## 1 Cultivate and harvest bacterial cells

Use 2–10 mL of a saturated E. coli culture (see page 8, table 3), pellet cells in a standard benchtop microcentrifuge
for 30 s at > 12,000 x g.
Discard the supernatant and remove as much of the liquid as possible.
> 12,000 x g, 30 s

## 2 Cell lysis

Add 150 μL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell
clumps remain before addition of Buffer A2!
Attention: Check Buffer A2 for precipitated SDS. If a white precipitate is visible, warm the buffer for several
minutes at 30–40 °C until precipitate is dissolved completely. Cool buffer down to room temperature (18–25 °C) before
use.
Add 250 μL Buffer A2. Mix gently by inverting the tube 5 times. Do not vortex to avoid shearing of genomic DNA.
Incubate at room temperature (18–25 °C) for up to 2 min or until lysate appears clear.
Add 350 μL Buffer A3. Mix thoroughly by inverting the tube until LyseControl has turned colorless throughout the
lysate without any traces of blue color. Do not vortex to avoid shearing of genomic DNA!
+ 150 μL A1
Resuspend
+ 250 μL A2
Mix
RT, 2 min
+ 350 μL A3
Mix

## 3 Clarification of lysate

Centrifuge for 3 min at full speed (> 12,000 x g).
> 12,000 x g, 3 min

## 4 Bind DNA

Place a NucleoSpin® Plasmid EasyPure Column into a Collection Tube (2 mL) and decant the supernatant from step 3 onto
the column.
Centrifuge for 30 s at 1,000–2,000 x g.
Discard flow-through and place the spin column back into the collection tube.
Load supernatant
1,000– 2,000 x g, 30 s

## 5 Wash and dry silica membrane

Add 450 μL Buffer AQ (supplemented with ethanol, see section 3).
Centrifuge for 1 min at full speed (> 12,000 x g).
Very carefully discard the collection tube and the flow- through and make sure the spin cup outlet does not touch the
wash buffer surface. Otherwise repeat the centrifugation step.
Note: To reduce ethanol carry-over to a minimum for better performance in downstream applications, incubate spin cup
for 10–15 min at 37 °C to dry silica membrane completely.
+ 450 μL AQ
> 12,000 x g, 1 min

## 6 Elute DNA

Place the NucleoSpin® Plasmid EasyPure Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 μL Buffer
AE.
Incubate for 1 min at room temperature (18–25 °C).
Centrifuge for 1 min at full speed (> 12,000 x g).
Note: For more efficient elution procedures and alternative elution buffer (e.g., TE buffer or water) see section
2.4.
+ 50 μL AE
RT, 1 min
> 12,000 x g, 1 min

Protocol 2 : NucleoBond® Xtra plasmid purification midi/maxi (Marchery Nagel)

## 1 Preparation of starter culture

Inoculate a 3–5 mL starter culture of LB medium with a single colony picked from a freshly streaked agar plate. Make
sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee plasmid propagation (see
section 4.3 for more information). Shake at 37 °C and ~ 300 rpm for ~ 8 h.

## 2 Preparation of large overnight culture

Inoculate an overnight culture by diluting the starter culture 1 / 1000 into the given volumes of LB medium also
containing the appropriate selective antibiotic. Grow the culture overnight at 37 °C and ~ 300 rpm for 12–16 h.
100 mL 300 mL

## 3 Harvest of bacterial cells

Measure the cell culture OD 600 and determine the recommended culture volume.
V [mL] = 400 / OD 600
V [mL] = 1200 / OD
600
Pellet the cells by centrifugation at 4,500–6,000 x g for ≥ 10 min at 4 °C and discard the supernatant completely.
Note: Optimal lysis conditions are achieved by a unique ratio of lysis buffer RES, LYS, and NEU to the cell mass. See
section 4.5 for recommendations concerning the optimal pelleted culture volume for cells containing high-copy
plasmids and section 4.6 for recommendations concerning cells with low-copy plasmids. Read section 4.5 carefully as
excess cell input will result in reduced yield. The recommended culture volumes below are calculated for a final OD
600 of around 4.

## 4 Resuspension (Buffer RES)

Resuspend the cell pellet completely in Resuspension Buffer RES + RNase A by pipetting the cells up and down or
vortexing the cells.
For an efficient cell lysis it is important that no clumps remain in the suspension.
Note: Increase RES buffer volume proportionally if more than the recommended cell mass is used (see section 4.8 for
information on optimal cell lysis and section 4.9 regarding difficult-to-lyse strains).

## 5

Cell lysis (Buffer LYS) !
Check Lysis Buffer LYS for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for
several minutes at 30–40 °C until precipitate is dissolved completely. Cool buffer down to room temperature (18–25
°C).
Add Lysis Buffer LYS to the suspension.
Mix gently by inverting the tube 5 times. Do not vortex as this will shear and release contaminating chromosomal DNA
from cellular debris into the suspension.
Incubate the mixture at room temperature (18–25 °C) for 5 min.
Warning: Prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate
contaminating chromosomal DNA into the lysate.
Note: Increase LYS buffer volume proportionally if more than the recommended cell mass is used (see section 4.8 for
information on optimal cell lysis).
8 mL 12 mL

## 6 Equilibration (Buffer EQU)

Equilibrate a NucleoBond® Xtra Column together with the inserted column filter with Equilibration Buffer EQU.
Apply the buffer onto the rim of the column filter as shown in the picture and make sure to wet the entire filter.
Allow the column to empty by gravity flow. The column does not run dry.
12 mL 25 mL

## 7

Neutralization (Buffer NEU) !
Add Neutralization Buffer NEU to the suspension and immediately mix the lysate gently by inverting the tube until
blue samples turns colorless completely! Do not vortex.
The flask or tube used for this step should not be filled more than two thirds to allow homogeneous mixing. Make sure
to neutralize completely to precipitate all the protein and chromosomal DNA. The lysate should turn from a slimy,
viscous consistency to a low viscosity, homogeneous suspension of an off-white flocculate. In addition, LyseControl
should turn completely colorless without any traces of blue.
Immediately proceed with step 8. An incubation of the lysate is not necessary.
Note: Increase NEU buffer volume proportionally if more than the recommended cell mass is used (see section 4.8 for
information on optimal cell lysis).
8 mL 12 mL

## 8

Clarification and loading !
Make sure to have a homogeneous suspension of the precipitate by inverting the tube 3 times directly before applying
the lysate to the equilibrated NucleoBond® Xtra Column Filter to avoid clogging of the filter.
The lysate is simultaneously cleared and loaded onto the column. Refill the filter if more lysate has to be loaded
than the filter is able to hold. Allow the column to empty by gravity flow.
Alternative: The precipitate can be removed by centrifugation at ≥ 5,000 x g for at least 10 min, for example if more
than double the recommended cell mass was used. If the supernatant still contains suspended matter transfer it to a
new tube and repeat the centrifugation, preferably at higher speed, or apply the lysate to the equilibrated
NucleoBond® Xtra Column Filter.
This clarification step is extremely important since residual precipitate may clog the NucleoBond® Xtra Column. To
load the column you can either apply the cleared lysate to the equilibrated filter or remove the unused filter
beforehand. Allow the column to empty by gravity flow.
Note: You may want to save all or part of the flow-through for analysis (see section 8.1).
Midi Maxi

## 9

1st Wash: Column filter and column !
(Buffer EQU)
Wash the NucleoBond® Xtra Column Filter and Nu-cleoBond® Xtra Column with Equilibration Buffer EQU.
Apply the buffer to the funnel shaped rim of the filter and make sure it is washing out the lysate which is remaining
in the filter. Omitting this step or just pouring the buffer directly inside the funnel may reduce plasmid yield.
5 mL 15 mL

## 10 Filter removal

Either pull out the NucleoBond® Xtra Column Filter or discard it by turning the column upside down.

## 11

2nd Wash: Column only (Buffer WASH) !
Wash the NucleoBond® Xtra Column with Wash Buffer WASH. It is important to remove the column filter before applying
the washing buffer to avoid low purity.
8 mL 25 mL

## 12 Elution (Buffer ELU)

Elute the plasmid DNA with Elution Buffer ELU. Collect the eluate in a 15 mL or 50 mL centrifuge tube (not provided).
Note: Preheating Buffer ELU to 50 °C prior to elution may improve yields for large constructs such as BACs.
Optional: Determine plasmid yield by UV spectrophotometry in order to adjust desired concentration of DNA in step 15
and calculate the recovery after precipitation.
5 mL 15 mL !
NucleoBond® Xtra Midi / Maxi Plus:
Proceed with step 13 for the centrifugation protocol after isopropanol precipitation or continue with section 7.3 for
plasmid concentration and desalting by using the NucleoBond® Finalizer (NucleoBond® Xtra Midi Plus) or NucleoBond®
Finalizer Large (NucleoBond® Xtra Maxi Plus).

## 13 Precipitation

Add room-temperature isopropanol to precipitate the eluted plasmid DNA.
Vortex thoroughly!
Centrifuge at ≥ 4,500 x g for ≥ 15 min at ≤ room temperature, preferably at 15,000 x g for 30 min at 4 °C. Carefully
discard the supernatant.
3.5 mL 10.5 mL

## 14 Washing and drying

Add room-temperature 70 % ethanol to the pellet.
2 mL 14 mL
Centrifuge at ≥ 4,500 x g, preferably ≥ 15,000 x g for 5 min at room temperature (18–25 °C).
Carefully remove ethanol completely from the tube with a pipette tip. Allow the pellet to dry at room temperature
(18–25 °C).
Note: Plasmid DNA might be harder to dissolve when over-dried.
10–15 min 15–30 min

## 15 Reconstitution

Dissolve the DNA pellet in an appropriate volume of buffer TE or sterile H2O.
Depending on the type of centrifugation tube, dissolve under gentle pipetting up and down or constant spinning in a
sufficient amount of buffer for 10–60 min (3D-shaker).
Determine plasmid yield by UV spectrophotometry. Confirm plasmid integrity by agarose gel electrophoresis.

Protocol 3 : 1% agarose gel

1. Measure 1 gram of agarose.
2. Add 100 mL of 1x TAE buffer.
3. Microwave for 1-3 min until the agarose is completely dissolved.
4. Let the agarose cool down to around 50 degrees celcius.
5. Add 100 µL GelRed, swirl the flask, and pour slowly into a geltray with well comb.
6. Let the gel solidify for 20 to 30 minutes at room temperature
7. Load DNA samples mixed with loading buffer / loading dye
8. Run at 80 to 150 V for 1 to 2 hours (dependent on gel percentage and voltage used)

Protocol 4 : NEB10-béta transformation protocol (New England Biolabs)

1. Thaw a tube of NEB 10-beta Competent E. coli cells on ice for 10 minutes.
2. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
3. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix
   cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture.
8. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
9. Warm selection plates to 37°C.
10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in
    NEB 10-beta/Stable Outgrowth Medium.
11. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate
    at 30°C for 24-36 hours or 25°C for 48 hours.

Protocol 5 : Restriction enzyme double digestion

1. Per 50 µL reaction mix the following:

* Up to 1 µg
* 5 µL 10x Cutsmart Buffer
* 10 units of restriction enzyme one
* 10 units of restriction enzyme two
* Fill reaction to 50 µL with nuclease free water

2. Incubate at 37 degrees Celsius for two hours to overnight
3. Inactivate enzymes by incubating for 20 minutes at 65 degrees Celcius

Protocol 6 : Chemocompetent cells

## Materials:

* Overnight Culture from the right bacteria.
* Tfb1, icecold: 30 mM natriumacetaat, 100mM rubidium chloride,10 mM calcium chloride, 50mM mangaan chloride, 15 %
  glycerol; pH 5,8 achieved by adding diluted NaOH. Sterilize by filter.
* Tfb2, icecold: 10 mM MOPS, 75 mM calcium chloride, 10 mM rubidium chloride, 15% glycerol; pH 6,6 achieving by
  adding diluted NaOH/ Sterilize by filter.
* Eppendorf cups
* Spectrofotometer
* Shaker at 37 degrees Celsius
* Centrifuge + tubes

## Day before making competent cells:

Put a bacteria into 2 ml LB medium at the beginning of the day. Shake at 125 rpm at 37 degrees celsius for at least 6
hours.
Dilute the culture 1:500 into 20 ml LB medium. (40 ul/20 ml). Shake at 125 rpm at 37 degrees celsius overnight.

## Day of making competent cells:

1. Dilute the overnight culture 1:100 into 200 ml LB medium (in a 1L flask). (2ml /200 ml)
   Shake the cells at 250 rpm and 37 degrees celsius till an OD600 of 0,5-0,7 is reached (this can take 2 hours).
   Measure every 30 minutes the OD600 with a cuvette and sterile LB medium as blanco.
2. When a OD600 between 0,5-0,7 is reached. Cool the cells down to 4 degrees celsius and centrifuge the culture for 10
   minutes at 5000 x g and 4 degrees celsius. The cells need to stay at ice from this step on as much as possible.
3. Dispose the supernatant and ad 66,4 ml cold TBF1 buffer /200 ml culture. Resuspended the pellet in the buffer
   completely.
   Centrifuge the culture for 10 minutes at 5000 x g and 4 degrees celsius.
4. Dispose the supernatant and ad 24 ml cold TBF1 buffer /200 ml culture. Resuspended the pellet in the buffer
   completely. Incubate the cells on ice for 15 minutes.
5. Centrifuge the culture for 10 minutes at 5000 x g and 4 degrees celsius.
6. Dispose the supernatant and ad 4 ml cold TBF2 buffer /200 ml culture. Resuspended the pellet in the buffer
   completely. Incubate the cells on ice for 20 minutes.
7. Divide the solution over eppendorf cups by pipetting 50 ul per cup. Code the cups and place the cups on dry ice for
   10 minutes. After fully frozen, store the cells at -80 degrees celsius.

Protocol 7 : Rapid DNA Dephos & Ligation

![](https://static.igem.org/mediawiki/2018/1/1b/T--Rotterdam_HR--Protocol7.png)

Protocol 8 : Digestion

1. For a double restriction enzyme digestion the following protocol was followed:
2. Per 50 µL reaction mix the following:

* DNA: Up to 1 µg
* Cutsmart Buffer(10x): 5 µL
* Restriction enzyme one: 10 units
* Restriction enzyme two: 10 units

3. Fill reaction to 50 µL with nuclease free water.
4. Incubate at 37 degrees Celsius for two hours or overnight, depending on which restriction enzymes you use.
5. Inactivate enzymes by incubating for 20 minutes at 65 degrees Celsius or by freezing the digestion solution for at least an hour by -20 degrees celsius.

Protocol 9 : Glycerol stock

* To make glycerol stocks a overnight culture was needed. The culture was made by putting a colony in LB-medium and incubate the culture for a night at 37ºC.
* After a night, the cultures are full enough and ready. To make a glycerol stock, 1 ml culture is mixed with 0,250 ml 87% glycerol solution. This is mixed by flicking the tube 3-5 times. The glycerol stock needs to be stored at -80ºC.
* When a glycerol stock is used, be sure that the stock is not fully thawed before putting it back at -80ºC.

Protocol 10 : Mediums

## LB medium

1. Mix 5 grams of Lithium Broth combination with 200 ml demi water. After full dissolving, the medium needed to be sterilised by heating it to 121 degree celsius for at least 30 minutes.
2. Store at 4 degrees Celsius :

## LB agar

1. Per 200 ml LB medium, 3 gram Agar-Agar was added and dissolved. After full dissolving, the medium needed to be sterilised by heating it to 121 degree celsius for at least 30 minutes.
2. Store at 4 degrees Celsius :

## SOC medium

1. The following protocol is for 1L SOC- Medium
2. Mix the following substances:

* 2% (m/v) tryptone
* 0,5% (m/v) yeast extract
* 10mM NaCl
* 2,5 mM KCl

3. Ad 980 ml demi water to this mixture. After full dissolving, the medium needed to be sterilised by heating it to 121 degree celsius for at least 30 minutes.
4. Make the following solutions:

* 10 mM MgCl2 (10 ml)
* 20 mM Glucose (10 ml)

5. Sterilize these solutions by filtering through a 0,22 um filter.
6. Add both solutions aseptic to the premade medium. Be sure the medium was cooled down before adding the MgCl2 and Glucose.
7. Store at 4 degrees celsius.

## TAE buffer

1. This protocol is for a 50x TAE buffer.
2. For use, we make a 1X solution by diluting it 50 times.

* Weigh out 242 grams of Tris-base (MW = 121.14 g/mol) and dissolve in approximately 700 milliliters of demi water.
* Carefully add 57.1 milliliters of 100 % glacial acid (or acetic acid) and 100 milliliters of 0.5 M EDTA (pH 8.0).
* Adjust the solution to a final volume of 1 liter.

3. The pH of this buffer is not adjusted and should be about 8.5.
4. Store stock solution at room temperature.

Protocol 11 : PCR

1. Materials: For our PCR (Both plasmid and colony) reactions we have used the standard neb protocols for a 25 μl reaction , with the following concentrations:

|  |  |
| --- | --- |
| 10X Standard Taq Reaction Buffer | 2.5 μl |
| 10 mM dNTPs | 0.5 μl |
| 10 μM forward Primer (VF2) | 0.5 μl |
| 10 μM reverse Primers (VR) | 0.5 μl |
||  |  |  |  |
| --- | --- | --- | --- |
| Taq DNA Polymerase | 0.125 ul |||  |  |
| --- | --- |
| Nuclease-free water | to 25 μl ||

2. Methods: A master mix is made with the concentrations shown above. In case of a plasmid pcr, a volume of plasmid is added before the reaction is filled with Nuclease-free water. With the colony PCR we used a toothpick to touch on the desired colony. Before briefly submerging and shaking the tip of the toothpick in the PCR Eppendorfcup filled with the mastermix. Before pricking it into a clean Agar plate a couple of times to grow more colonies in case the PCR shows us the desired products

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Rotterdam HR/Results

# Results

# Results

# General

![](https://static.igem.org/mediawiki/parts/5/5e/T--Rotterdam_HR--foto1Res.jpeg)
Our plan was to create multiple composite parts, which included CooA.
To be pure the DNA needed to have a A260/280 value between 1,80-2,00 and a A260/230 value between 2,00 and 2,20.
These composite parts would have different
promoters and output genes, so we can measure carbon monoxide in multiple ways.

For further steps we isolated the DNA. The DNA had to be pure for digestions. To be sure the DNA was pure,
requirements were set.
Hereby there would be looked at the curve of the line also. If the line wasn’t smooth, the DNA was most likely not
pure. A pure sample has a curve like shown in figure 1.

To produce enough DNA of BBa\_J04450 (an plasmid containing a functional spacer) we performed multiple mini preps
(DNA
isolations), which gave us the following results:

|  |  |  |
| --- | --- | --- |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 1 | 270,71 ng/µL |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 2 | 119,84 ng/µL |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 1 | 451,17 ng/µL |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 2 | 319,01 ng/µL |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 1 | 136,98 ng/µL |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) 2 | 80,90 ng/µL |

Successful isolated DNA could be digested and ligated with another biobrick for testing. Early named biobricks were
paired with each other, where BBa\_K133071 and BBa\_K173003 functioned as vectors and BBa\_I134353 and BBa\_J23100 as
inserts. We used the backbone from the vectors, so we only needed to insert 1 biobrick into another biobrick. To
know
if the ligation had worked the DNA was transformed into the E.coli strain: NEB10Bèta. Colonies that grow were
tested by digesting again. The digested DNA was put on gel, and the length was determined.

# CooA production

When we started our project our first order of business was to reliably produce CooA, the carbon monoxide dependent
transcription factor. We knew we had to constitutively produce CooA, and had to produce as much as we could to
elicit
a high enough response, but we also knew that our cells wouldn’t be able to produce it indefinitely. To this end we
decided to combine different constitutive promoters (BBa’s [J23100](http://parts.igem.org/Part%3ABBa_J23100),
[J23105](http://parts.igem.org/Part%3ABBa_J23105) and [J23113](http://parts.igem.org/Part%3ABBa_J23113)),
and combine them with
different ribosomal binding sites (BBa’s [B0030](http://parts.igem.org/Part%3ABBa_B0030), [B0031](http://parts.igem.org/Part%3ABBa_B0031)
and [B0032](http://parts.igem.org/Part%3ABBa_B0032)). This could then be combined with CooA
([BBa\_K352001](http://parts.igem.org/Part%3ABBa_K352001)) and a double terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)).

At first we tried to insert our ribosomal binding site into the plasmids with the different promoters, though
because
the backbones of the promoters were different from the standard registry backbones we had some difficulty with the
cloning, so we decided to turn our plan around and tried to insert the promoters into the plasmids containing the
different ribosomal binding sites.
The next problem we had with our cloning however was the size of the promoters themselves. As they are only around
15
base pairs long, and the ribosomal binding sites are around 35 base pairs long, when combined the total change in
plasmid size would only be around 50 to 60 base pairs, which we couldn’t visualize on our electrophoresis gels.

After this conundrum we had decided to follow the standard iGEM 3A assembly method, using [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450)
([pSB1C3](http://parts.igem.org/Part%3ApSB1C3),
[pSB1K3](http://parts.igem.org/Part%3ApSB1K3) and [pSB1A3](http://parts.igem.org/Part%3ApSB1A3)
versions of this biobrick) as a quick screening method.

![](https://static.igem.org/mediawiki/parts/4/4f/T--Rotterdam_HR--foto2Res.jpeg)
After a few attempts we seemed to have successfully cloned [BBa\_J23105](http://parts.igem.org/Part%3ABBa_J23105)
with [BBa\_B0032](http://parts.igem.org/Part%3ABBa_B0032) and [BBa\_K352001](http://parts.igem.org/Part%3ABBa_K352001)
with [BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015).
This has then been cloned together to make the full CooA production plasmid. Of the resulting colonies we made 10
different colony PCR’s, and put these on gel, of which one seemed to be the expected size.

Sadly when we sequenced the CooA part in pSB1A3, the sequence conformed to the sequence of [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450),
which is about the same length as the expected CooA product from the PCR.

# Gas production

For our gas production system we started with transformations of the biobricks:

-BBa\_J23100: Constitutive promoter.

-BBa\_K133071: UreB.

-BBa\_K173003: Pyruvate decarboxylase + ethanol dehydrogenase.

-BBa\_I13453: Arabinose Promoter.

Things went wrong, so after a time we had transformed the biobrick combinations BBa\_J23100 + BBa\_K133071, BBa\_J23100
+ BBa\_K173003, BBa\_I13453 + BBa\_K133071 and BBa\_I13453 + BBa\_K173003 in the E.coli strain NEB10Bèta.

After the transformations in NEB10bèta we started testing with our self invented gas production test tubes.

![](https://static.igem.org/mediawiki/parts/8/8d/T--Rotterdam_HR--foto3Res.jpeg)
![](https://static.igem.org/mediawiki/parts/3/31/T--Rotterdam_HR--foto4Res.jpeg)

Due to us not having any experience with our setup as seen in these last two images, when we placed the smaller tube
into a 15 mL
tube there was some gas present. Though when comparing before and after pictures there is undoubtedly more gas
present in our tubes.

![](https://static.igem.org/mediawiki/parts/6/67/T--Rotterdam_HR--foto5Res.jpeg)
![](https://static.igem.org/mediawiki/parts/d/d2/T--Rotterdam_HR--foto6Res.jpeg)

| Bacteria | LB | LB | Urea | Urea | Pyruvate | Pyruvate |
| --- | --- | --- | --- | --- | --- | --- |
| NEB10beta | + | +++ | +++ | +++ | + | ++/+++ |
| BL21 (DE3) | - | ++ | - | - | - | - |
| BL21 | - | - | - | - | - | - |
| DH5a | +++ | +/- | - | - | +/- | + |
| HB101 | - | - | - | - | + | - |
| JM109 | - | - | - | - | - | - |

-: No/ minimal gas production,
+: Little bit of gas production
++: More than a little bit of gas production
+++: Most gas production

The strains are tested with different substrates in Lysogeny Broth (LB) culture medium (See table 2). HB101 and JM109
can be used best as a negative control for our gas production tests.

For easier use we gave our combinations abbreviations. This can be seen in the following tabel:

| Number | Promotor | Gene | Backbone |
| --- | --- | --- | --- |
| A | K352002 (CooF) | K173013 | pSB1K3 |
| B | K352002 (CooF) | K173003 | pSB1K3 |
| C | K352002 (CooF) | K133071 | pSB1K3 |
| D | K352003 (CooM) | K173003 | pSB1K3 |
| E | K352003 (CooM) | K173013 | pSB1K3 |
| F | K352003 (CooM) | K173013 | pSB1K3 |
| H | I13453 | K173013 | pSB1K3 |
| I | I13453 | K173003 | pSB1K3 |
| J | I13453 | K133071 | pSB1K3 |

![](https://static.igem.org/mediawiki/parts/d/d7/T--Rotterdam_HR--foto7Res.jpeg)
![](https://static.igem.org/mediawiki/parts/b/bd/T--Rotterdam_HR--foto8Res.jpeg)

When looking at figures 6 and 7 we can conclude that most biobricks did not give the expected results, because they
were cut twice. H1, D2, I2 and J2 were digested once,so we expect they include the right biobricks and BBa\_K352002 +
BBa\_K133116 and BBa\_K352003 + BBa\_K133116 were dropped because those lanes are empty.

![](https://static.igem.org/mediawiki/parts/a/a2/T--Rotterdam_HR--foto9Res.jpeg)
![](https://static.igem.org/mediawiki/parts/6/61/T--Rotterdam_HR--foto10Res.jpeg)

Only I1, J1 and J2 gave usable results so we sequenced all parts that we couldn't say were definitely wrong.

# Sequencing and shipping

We sequenced the previously mentioned gas production parts and CooA 8 (refer to figure: 2). When comparing the
sequencing results to the expected sequences we concluded that B1, B6, D1, I1 and J1 were as we expected.
These BioBricks were then digested and ligated into pSB1C3, after which we transformed them, mini prepped them, and
dried the DNA onto a 96 wells plate for submission.

![](https://static.igem.org/mediawiki/parts/1/10/T--Rotterdam_HR--foto11Res.jpeg)
![](https://static.igem.org/mediawiki/parts/d/d5/T--Rotterdam_HR--foto12Res.jpeg)
![](https://static.igem.org/mediawiki/parts/2/24/T--Rotterdam_HR--foto13Res.jpeg)
![](https://static.igem.org/mediawiki/parts/d/d2/T--Rotterdam_HR--foto14Res.jpeg)
![](https://static.igem.org/mediawiki/parts/0/0a/T--Rotterdam_HR--foto15Res.jpeg)
![](https://static.igem.org/mediawiki/parts/8/87/T--Rotterdam_HR--foto16Res.jpeg)
![](https://static.igem.org/mediawiki/parts/a/af/T--Rotterdam_HR--foto17Res.jpeg)
![](https://static.igem.org/mediawiki/parts/9/9c/T--Rotterdam_HR--foto18Res.jpeg)

# Hardware

For information about our hardware results, please navigate to the Hardware page.

[To the hardware page](https://2018.igem.org/Team%3ARotterdam_HR/Hardware)

# Future plans

If we want to make a fully usable product firstly we would have to finish our CooA production plasmid. Secondly we
would like to look at a cell-free system, probably by altering the CooA structure, to avoid regulations regarding
GMOs outside of a laboratory setting. This would also mean a relatively simpler hardware design, cutting down costs
and the amount of work. If a cell-free system is not feasible we could also give more thought to safety measures,
adding a working kill switch to our system, and also pursuing possible hardware contingency plans (like releasing
toxic substances in our system when part of it breaks).

# Replicating the experiments

If we would do our experiments again, we would start the change by performing our digestions in a smaller end volume,
so we can get a more concentrated digest product, enabling us to use more nanograms of DNA when performing our
ligation. Secondly we would like to sequence our intermediate constructs, as most parts we wanted to use are fairly
small, and thus they don’t make a significant enough change in construct length to easily and reliably detect on an
electrophoresis gel. So as to be sure that our intermediate constructs are as we want them to, sequencing would be
the most reliable method.