Team:Rotterdam HR/Notebook
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Blue White screening on paper
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July 21
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
July 23
ATP sensor
July 24
Gas output
July 24
Notebook generator
July 24
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.
Transformation
July 24
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
July 24
Notebook generator
July 25
Google Drive API
July 25
Automatic uploader
July 25
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.
Transformation
July 27
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
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
July 27
Automation program
July 29
Automatic uploader
July 29
Page generator
July 31
Google Drive API
July 31
Automation program
July 31
Automation program
August 1
Notebook
August 2
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.
Transformation
August 9
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 10
Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.
Urea and sodium pyruvate test for resistence E.coli
August 10
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 13
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 13
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 13
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.
Testing gas production
August 16
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.
Testing gas production
August 23
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.
Testing gas production
August 23
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.
Testing gas production
August 23
Minipreps are made of colonies 11 and 19 of biobrick combination K133071 + J23100. Results: 11. 270,57 ng/ul 19. 253,59 ng/ul.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 27
A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
August 27
Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.
Urea and sodium pyruvate test for resistence E.coli
August 28
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 7
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 7
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 11
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.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 11
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/�l and for 2: 355,3 ng/�l.
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 17
Cloning Multiple BioBricks to Assess Gas Production BioBricks in E.Coli
September 17
Different species/tribes? of E.coli are tested as negative control. Those spacies/tribes? 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 tribe? 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 tribes.