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 be&#769ta 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&degC.

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

After making a 20 mL overnight culture of our NEB 10 be&#769ta cells containing our BioBricks, we performed a mini prep (Protocol 3). Using our nanodrop spectrophotometer  which gave us the following DNA concentrations:
 

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. 

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. 

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. 

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. 

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

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.

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. 

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

Transformations of the biobricks (K133071 + J23100), (K13071 + I1345), (K173003 + J23100) and (K173003 + I13453) in NEB10B�ta. There was no grow except for the biobrick combination K133071 + J23100.

A colony PCR is done for the NEB10B�ta 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.

The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 has 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. 

Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.

Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453) and a negative control.

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.

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.

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

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

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

Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promotors (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 NEB10b�ta and digested again as control.

Transformations of the biobrick combinations in NEB10b�ta. 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.

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.
 

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.

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

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. 

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.

Different strains of E.coli are tested as negative control. Those strains were: NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha and JM109. NEB10B�ta 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.

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.

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.

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.

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. After this we started with PCR.

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.

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

Table 1: Overnight culture scheme.