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

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)

Today we performed a digestion, dephosphorilation and ligation (following protocols 4 and 5) of a few promotor and RBS BioBricks. Because of administrative compliations only the variants with J23113 and a RBS could be made.

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

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.

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

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.

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.

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.

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.

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.

We did another round of plasmid isolation for stocking.

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

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. 
 

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

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

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. 

 

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 

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 

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

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 Michels 
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

Method: 
Miniprep protocol...??
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.....???

Results

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

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. 

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 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

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.

 

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.
 

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.

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

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

 

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
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

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

15-08-18
Gas was let into the little tubes so it was difficult to see whether there was gasproduction or not. Mainly in the negative control this was 

 

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.

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.

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.

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

 

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
See experiment 3

Methods 
See experiment 3

Results

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

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

Experiment 9: Minipreps
28-08-18

Materials
See experiment 3

Methods
See experiment 3
Make minipreps of the biobricks K352002, K352003

Results
See figure 1

29-08-18
Materials
See experiment 3

Methods
See experiment 3
Make minipreps of the biobrick K352002

Results
See figure 2

30-08-18
Materials
See experiment 3

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

Results
See image experiment 9

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.
 

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
See experiment...

Methods
See experiment ..

 

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.
 

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: transformation of the biobrickombinations 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.
 

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
 

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.

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

 

 

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
See experment ...

Methods
See experiment ...

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, 

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	-	-	-	-	-	-

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
See experiment 3

Methods
See experiment 3

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

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

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
See experiment ...

Methods
See experiment ...
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. 

 

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.
 

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.
 

Gelelectrophoresis
01-10-18


02-10-18
Gelelectrophoresis


03-10-18
Gelelectrophoresis

Gel 1
L, D2.1, D2.2, D2.3, D2.5, D2.7, D2.8, D2.10, D not digested, L, J3.1, J3.2, J3.3, J3.5, J3.7, J3.8, J3.10, J not digested, L.

Gel 2
L, H2.1, H2.2 H2.4, H2.6, H2.7, H2.9, H2.11, H2 not digested, L, I2.1, I2.2, I2.4, I2.6, H2.7, H2.9, H2.11, H2 not digested, L

Gel 3
L, C1.1, C1.2, C1.4, C1.6, C1.7, C1.8, C1.10, C1 not digested, L, C2.1, C2.2, C2.4, C2.6, C2.7, C2.8, C2.10, C2 not dgested, L

Gel 4
L, C3.1, C3.2, C3.4, C3.6, C3.7, C3.8, C3.10, L, C4.1, C4.2, C4.4, C4.6, C4.7, C4.8, C4.10, C4 not digested, L

Gel 5
L, C6.1, C6.2, C6.4, C6.6, C6.7, C6.8, C6.10, C6 not digested, L, B5.1, B5.3, B5.4, B5.4 (other tube), B5.6, B5..., B5.9, B5.11, 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
Gel 2

Gel 3

Gel 4

Digestions with EcoRI-HF and PstI are done for the biobrick numbers A2, B1, B5, B6, D1, E1, E3, I1, J1, J1.2, CooA (8) and pSB1C3. This is done because now the Kanamycin backbone can be replaced for the Chloramphenicol backbone. This is done by dephosphorylation and ligation.

Experiment 24: Digestions, gelelectrophoresis and dephosphorylation and ligation.
08-10-18 <---- klopt dit?

Materals
See experiment ...
-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

Table 1: Master mix

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 scheme
.........

Results
See figure 1.

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

Number	HB101	JM109
J1	1x	1x
I1	3x	3x
D1	2x	2x
B1	1x	1x
B6	1x	1x

Table 1: Overnight culture scheme.

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	HB101	JM109
J1	1x	1x
I1	3x	3x
D1	2x	2x
B1	1x	1x
B6	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