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 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.

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.

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.
 

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.

 

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.

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.

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.

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 

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. 

 

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

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 

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.

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.

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
 

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).

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.

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

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!

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.

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
 

Repeated transformation.

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

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
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

 

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

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.

 

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.

 

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

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.

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

 

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.
 

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

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. 

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.
 

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.
 

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
 

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.

 

 
 

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.

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

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
-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

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	-	-	-	-	-	-

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).


 

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

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
 

 
 

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. 

 

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.
 

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.

 

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

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

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