TeamSlovenia HS Lab&Safety Notebook.html

Team Slovenia_HS: Notebook

Notebooks



Our Journey

 

''If you want to go quickly, go alone. If you want to go far, go together.''


- African proverb -

  The beginning of our journey

 

When we first saw that The National Institute of Chemistry was looking for team members to present the colours of Slovenia iGEM HS Division, we thought it would be a great opportunity to do what something we were interested in and learn something new. We applied to the call for applications and hoped for the best. Parts of the application procedure were writing a motivational letter, attending an interview, taking part in a personality test and, the toughest of them all, solve a scientific problem.
 14 months ago, we knew nothing about synthetic biology because we only learned the basics in our biology and chemistry classes. We had no clue how demanding the project is going to be at the time.

Building a knowledge basis

 

When we started with our team meetings in September and October 2014,  we first had to learn the basics of biochemistry and biotechnology.  Through autumn and winter we have had having lessons two afternoons per week after school. We learned about cloning, plasmids, PCR and biobricks. Moreover, we learned how to edit wiki pages, use bioinformatics programs and data bases. At the same time we were also studying the additional literature and developing our ideas. We learned about E. coli, conditions for its optimal growth and bioreactors. Finally, we got to enter the labs where we practiced basic lab techniques and protocols, such as pipetting with micropipettes. We also cut the vectors with restriction enzymes for the first time and tested our samples using agarose gel electrophoresis.  It needless to say how fascinated we were when we were actually able to see several pieces of DNA of different lengths.  It was a great experience because we were tutored by university professors and scientists who were really able to explain to us everything thoroughly.

So Let’s get to work!

 

Afterwards we decided to divide in two working groups: biotechnologists and synthetic biologists. Biotechnologists worked under the supervision of The National Institute of Chemistry while synthetic biologists worked under the supervision of The Faculty of Chemistry and Chemical Technology. We also held regular team meetings during which we organized our teamwork and exchanged our results and experience.
  When we first actually started working in the lab we wanted to obtain the genes needed for building our construct from a mixed culture which we knew also contained Clostridium. We wanted to do so because The Laboratory for Environmental Sciences and Engineering already used this culture for their own experiments and we wanted to make use of it as well. As our DNA purification from mixed culture was unsuccessful we decided to go a step further and order pure culture and purify the DNA out of it. However, this was also unsuccessful and we decided to order synthetic DNA segments from IDT Technologies. These arrived in late June and since then we spent more or less every day in the lab.

  Summer of tears and sweat (and empty AGE gels)

We spent the following months sweating in the lab. Our days consisted of cloning, doing colony PCRs, testing our samples, hoping for positive results of agarose gel electrophoresis and crying as this was rarely the case. At the same time we prepared different mediums to see what best suits for E. coli's growth, measured absorbance, worked in both aerobic and anaerobic conditions (the following were especially fun), carried out the ecotoxicological tests, isolated n-butanol, prepared different mediums again and analysed tons of results.

There is life outside the lab too…

 

Besides the lab work we also filmed a short clip about our project and presented our project and principles of synthetic biology to a wider audience. We met the Slovenian prime minister while doing so and made sure he understood what we were trying to accomplish. We established our very first crowdfunding campaign on Indiegogo, wrote articles, collaborated with other iGEM teams, searched for sponsors and designed our wiki.

The lessons we learned

To be honest, we have never imagined how demanding it would be to manage such a project. In fact, it turned out to be much more than just a scientific research. Being a part of this project was a great opportunity to experience real-life scientific teamwork, develop organisational and leadership skills. On the other hand we had to overcome a lot of obstacles, from having troubles with bureaucracy to not being able to get our vectors into bacteriae. It was then when we realized only we are the ones responsible for the project and that it is on us to reach out for help. We learned that in the end you always have to do what you have to do, no matter how hard it may seem. Perhaps the crucial lesson of this summer was about the importance of collaboration with others or, to put it another way, to stick together through thick and thin.

Notebook of construction

1.4.2015

•DNA cloning with lyophilized bacteria
•Our matrix were 2 mixed culture of lyophilized bacteria cells (from National Institute of Chemistry) which were resuspended in water and sonified.
•We attempted to clone 2 genes:
CtfAB and BdhA

2 concetnrations of each culture were prepared:
•original concentration
•10x dilluted
•PCR of cultures
•Agarose gel electrophoresis, PCR was not successful

2.4 2015

•DNA extraction from sludge.

8.4.2015

•DNA cloning with isolated DNA
•Our matrix was DNA previously isolated from active sludge and the active sludge itself.
Two concentrations of isolated DNA were prepeared:
•the original concentration
•200x diluted

We attempted to clone the following genes:
•BdhB
•BdhE2
•AdhE
•CtfAB
•BdhA

We tried to optimise PCR by varying the annealing temperature of primers: 40, 45, 50 and 55 °C.

22.4.2015

•Gene isolation from clostridium acetobutylicum We ordered new specific oligonucleotides (BdhA, CtfAB).

•Our goal was the isolation of genes reffering to Clostridium acetobutylicum.

As oligonucleotides we have used BdhA (R), Bdha (F), ctfAB (R), ctfAB (F), 16S (F), 16S (R).
Isolation sources:
• broth
• 10x diluted broth
• DNA
• 100x diluted DNA
PCR (master mix preparation) Agarose gel electrophorsis preparation AGE: 1- broth, 2-100x diluted broth, 3- DNA, 4- 100x diluted DNA We can see that there are some of the products, but they do not correspond of all the masses of the products. CfAB and BdhA are both 1000 bp long. 16S is 600 bp long. The reaction has to be repeated.

6.5.2015

•DNA cloning with pure culture

•Our matrix was pure Clostridium acetobutylicum culture. Two concentrations were prepeared:
•the original concentration
•50x diluted
We attempted to clone the following genes:
•16S
•CtfAB
•AdhE
•AdhE2
•BdhA
•BdhB
Agarose gel electrophoresis showed that only 16S was successfully cloned.

Only 16S was successfully cloned.

7.5.2015

DNA cloning
Our matrix was pure Clostridium acetobutylicum. A fresh master mix for cloning the following genes was prepared for:
•16S •CtfAB •AdhE •AdhE2 •BdhA •BdhB

Agarose gel electrophoresis showed that only 16S was successfully cloned.

20.5.2015

•iGEM transformation efficiency kit test

28.5.2015

•preparation of transformed E. coli for negative controls.
Constructs:
•promotor + RBS
•only terminator

22. 6. 2015

•Ligation with pJET 1.2 VECTOR, overnight incubation, PCR

24.6.2015

•Isolation of plasmid DNA from liquid medium via protocol.

30. 6. 2015

•inoculation of the negative controls into liquid LB media, DNA purification (AdhE1 and AdhE2), preparation of cloning vectors (with inserts BdhA, CtfA, CtfB) and their transformation into E. coli

1.7. 2015

•colony PCR (transformed cells with BdhA, CtfA, CtfB)

2.7.2015

•Agarose gel electrophoresis
•Colony PCR of samples

•We were repeating agarose gel electrophoresis
•unsccessful gene isolation from sludge due to Electrophoresis for CtfA was succesfull
•Plasmid DNA isolation using centrifuges

3. 7. 2015

•AGE of yesterday's samples, DNA purification (CtfA), colony PCR (BdhA, CtfB)

6. 7. 2015

•AGE of the last colony PCR samples, DNA purification (CtfB), colony PCR (BdhA), AGE of the samples (colony PCR of BdhA insert), BdhA ligation and transformation repeated

7. 7. 2015

•AGE of the samples (colony PCR with BdhA insert of the yesterday's transformed cells). Colony PCR repeated with new primers and new master mix. Inoculation of the transformed cells (with BdhB, AdhE1, AdhE2, CtfB, CtfA inserts) into liquid LB media. Restriction of the purified DNA (plasmids with CtfA, CtfB, BdhB, AdhE1 and AdhE2 inserts).

8. 7. 2015

•AGE of the samples (last colony PCR and restriction samples). DNA purification repeated (BdhB, AdhE1, AdhE2, CtfA, CtfB)

13.7.2015

•Plasmid DNA isolation using centrifuges
•measuring of absorbance of constructs
•Control restriction of genes, linearization AGE of restrictions

20. 7. 2015

•colony PCR of CtfB, BdhB and CtfA cells, AGE of the samples. Inoculation of E. coli with BdhB, CtfB, AdhE2, CtfA plasmids and an empty pSB1c vector in a liquid LB media

21.7.2015

•CtfA, CtfB, BdhB restriction with Xba1 and Pst1
•Restriction mixture was incubated at 37°C for 3h. PCR of samples: markings: 1-10 CtfA, 11-20 CtfB, 21-30 BdhB Agarose gel electrophoresis of PCR samples

Expected lenghts of genes:
•CtfA: 735 bp
•CtfB: 744 bp
•BdhA: 1250 bp
•AdhE1: 1666 bp
•AdhE2: 1020 BP

Electrophoresis was successful for the restriction products. Fragments with appropriate lenghts were isolated from the gel.

•DNA gel extraction
•Ligation
•overnight incubation on 37°C.
These fragments were then inserted into the expression vector pSB1C3.

22.7.2015

Transformation of Plasmid pSB1C3 into cells E.Coli DH5α was conducted accordingly to Heat shock

3.8.2015

•Agarose gel electrophoresis of PCR samples
•Pouring of plates, 400 mL
•Transformation of (35, 43, 51) appropriate samples , smearing on the plates (200 µL)
•Inoculation of transformed cells
•Measuring of absorbance

4.8.2015

•Plasmid DNA isolation using centrifuges (CtfB, BdhB)
•Measuring of absorbance
•Inoculating on liquid medium
•Protein isolation (CtfB, BdhB)
•Restriction for pSB1C3+P+RBS+CtfB+dT plasmid (with Spe1, Pst1) and pSB1C3+P+RBS+BdhB+dT plasmid (with Xba1, Pst1)
•Overnight incubation on 37 °C

23. 7. 2015

colony PCR of the transformed cells, AGE of the samples, colony PCR and AGE repeated

24. 7. 2015

restrictions of vectors
CtfA forward insert
CtfB forward insert
BdhB forward insert
CtfA back insert
CtfB back insert
BdhB back insert
negative control 1
negative control 2
AGE of samples
DNA purification out of gel
ligation into pSB1c
transformation of cells

25. 7. 2015

colony PCR of yesterday’s samples
repeating the yesterdays’ unsuccessful restrictions
AGE of the samples
DNA purification out of gel
ligation

26. 7. 2015

•colony PCR of yesterday’s samples
•repeating the yesterdays’ unsuccessful restrictions
•AGE of the samples
•DNA purification out of gel

27. 7. 2015

•ligation of yesterdays’ samples into pSB1c
•transformation of the cells

30. 7. 2015

•DNA purification using kit
transformation of AdhE1 and AdhE2 plasmids into the cells
restrictions
•CtfA forward insert
•CtfB forward insert
•Bdhb forward insert
•negative control 1 back insert
•AdhE1
•AdhE2

31. 7. 2015

•colony PCR of the samples
•DNA purification using kit
•ligation of negative controls

5.8.2015

•CtfA gene grew
•Plasmid isolation using centrifuges
•AGE restrictions
•Measuring of absorbance
•Agarose gel electrophoresis
•Ctf
•Antibody incubation
•Ligation mixture (CtfB, BdhB)
•Restriction of CtfA and CtfB
•Overnight incubation on 37 °C

6.8.2015

•Inoculating of constructs CtfB and BdhB on plates.
•Measuring of absorbance
•Isolation of proteins (CtfA, CtfB and BdhB)

7.8.2015

•Restriction of CtfA(Spe1), CtfB (EcoR1)
•Adding CtfB, BdhB in AGE gel.
•Measuring of absorbance

10.8.2015

•Isolation of proteins

11.8.2015

•Colony PCR: 35 (CtfA), 43 (CtfB), 51 (BdhB)

12.8.2015

•AGE of constructs (62 samples)
•Western blot
•Restriction of pSB1C3 with EcoR1, Pst1 for biobricks
•Overnight incubation on 37 °C
•Inoculating CtfA, CtfB, BdhB on liquid medium
•Transformation in pJET

13.8.2015

•Agarose gel electrophoresis (CtfA, CtfB, BdhB, pSB1C3)
•Plasmid isolation using centrifuges (Pjet), inoculating, because of unsuccessful growth
•Colouring of proteins
•Transformation of BdhB in Pjet.

14.8.2015

•Colouring of proteins (CtfA, CtfB)
•Protein electrophoresis
•Plasmid isolation using centrifuges (BdhB, CtfB)
•Inoculating on medium (CtfA, CtfB, BdhB)

18.8.2015

•Colony PCR for AdhE in pJET.
•Agarose gel electrophoresis
•Transformation with ligations PCR with AdhE1
•Transformation

19. 8.2015

•colony PCR and AGE
•1-10 pSB1c3+ctfA
•11-20 pSB1c3+ctfB
•21-30 pSB1c3+bdhB
•31-40 pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt
•R: Unsuccesful
•colony PCR and AGE
•1-30 pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt
•31 pSB1c3+prom+RBS+ctfA+dt
•32 pSB1c3+ prom+RBS+ctfB+dt
•R: only ctfB is succesful

20.8.2015

•restriction Adhe1 and Adhe2 with Kpnl, AGE, isolation from gel and restriction with Apal
•R: Apal doesn't cut well
•restriction of pSB1c3+prom+RBS+ctfA+dt(BV) and pSB1c3+prom+RBS+ctfB+dt (BI) over night

21. 8. 2015

•restriction of pSB1c3+prom+RBS+ctfB+dt(FV) and pSB1c3+prom+RBS+ctfB+dt (FI)
•AGE of the restrictions for forward ligation and restrictions for back ligation from yesterday
•R: unsuccesful

24. 8.2015

•cross cleveage (cutting each plasmid with only one restriction enzyme instead of double digest) with samples. The samples are cut with one restriction enzyme and only a part of the sample is loaded on gel. If the restriction is succesful, the other restriction enzyme (in parenthesisI will be added to complete the restriction
•pSB1c3+prom+RBS+ctfA+dt with Spel (EcoRl)
•pSB1c3+prom+RBS+ctfA+dt with EcoRl (Spel)
•pSB1c3+prom+RBS+ctfA+dt with Spel (Pstl)
•pSB1c3+prom+RBS+ctfA+dt with Pstl (Spel)
•pSB1c3+ prom+RBS+ctfB+dt with Xbal (Pstl)
•pSB1c3+ prom+RBS+ctfB+dt with Pstl (Xbal)
•pSB1c3+ prom+RBS+ctfB+dt with Xbal (EcoRl)
•pSB1c3+ prom+RBS+ctfB+dt with EcoRl (Xbal)
•pSB1c3+prom+RBS+ctfB+dt with Spel (EcoRl)
•pSB1c3+prom+RBS+ctfB+dt with EcoRl (Spel)
•pSB1c3+prom+RBS+ctfB+dt with Spel (Pstl)
•pSB1c3+prom+RBS+ctfB+dt with Pstl (Spel)
•pSB1c3+ prom+RBS+bdhB+dt with Xbal (Pstl)
•pSB1c3+ prom+RBS+bdhB+dt with Pstl (Xbal)
•pSB1c3+ prom+RBS+bdhB+dt with Xbal (EcoRl)
•pSB1c3+ prom+RBS+bdhB+dt with EcoRl (Xbal)
R: restrictions with the first enzyme were succesful, so the second enzyme was added over night

25. 8.2015

•we have new competent cells, so we are repeating Transformation
•Efficency Test
R: no colonies grew
•AGE of yesterday's restrictions
R: mostly unsuccesful, we isolate only pSB1c3+prom+RBS+ctfB+dt as back insert (BI)
R: unsuccesful

26. 8.2015

•Repeating the cross cleveage with the same samples, AGE, adding the second enzyme over night
•Repeating the Transformation Efficency Test
R: succesful

27. 8.2015

•AGE of yesterday's restrictions R: succesful, we isolate pSB1c3+prom+RBS+ctfA+dt (BV), pSB1c3+prom+RBS+ctfB+dt (BI), pSB1c3+prom+RBS+ctfB+dt (BV), pSB1c3+prom+RBS+bdhB+dt (BI), pSB1c3+prom+RBS+ctfB+dt (FI), pSB1c3+prom+RBS+bdhB+dt (FV)
•ligation for 2 hours at room temperature and transformation (for eachmixture we transform 2 tubes of cells, one with 2 ul, the other with 6 ul of ligation mixture; the rest of ligation mixture is left over night at 16
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI) pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI) prom+RBS+ctfB+dt (FI) ligated with pSB1c3+prom+RBS+bdhB+dt (FV) R: the next day nothing grew

28. 8.2015

•as the cells transformed with ligation mixture did not grow, we again transform new cells with the same ligation mixture that was left on 20 °C over night
•SDS-page of the cultures pSB1c3+prom+RBS+ctfA+dt, pSB1c3+prom+RBS+ctfB+dt, pSB1c3+prom+RBS+bdhB+dt that were grown for 3 days on 16 °C in hopes that the produced proteins would appear in the soluble fraction
R: all genes appear to be active, however they remain in the unsoluble fraction

31. 8.2015

•we recieved constructs from Aalto Helsinki (pSB1c3+GFP), which we transform the cells with
•transformation of cells with the biobrick BBa_K863010 (lacase) and an empty vector pSB1c3 as control
restriction of:
•pSB1c3+prom+RBS+ctfA+dt as BV, FI
•pSB1c3+prom+RBS+ctfB+dt as BI, BV, FI, FV
•pSB1c3+prom+RBS+bdhB+dt as BI, FV
R: forward vectors and inserts were not succesful, but back inserts and vectors were
we isolate from the gel:
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+prom+RBS+ctfB+dt as BI, BV
•pSB1c3+prom+RBS+bdhB+dt as BI
•we prepare the ligation for 2 hours at room temperature and then transform the cells pSB1c3+prom+RBS+ctfA+dt (BV) ligated with +prom+RBS+ctfB+dt (BI) pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI)
R: the next day nothing grew

1. 9.2015


restriction and AGE of:
•pSB1c3+prom+RBS+ctfA+dt as BV, FI
•pSB1c3+prom+RBS+ctfB+dt as BI, BV, FI, FV
•pSB1c3+prom+RBS+bdhB+dt as BI, FV
R: except for pSB1c3+prom+RBS+ctfA+dt (FI) the restrictions were succesful
We isolate from the gel:
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+prom+RBS+ctfB+dt as BI, BV, FI
•pSB1c3+prom+RBS+bdhB+dt as BI, FV
•we set the ligation on 16 °C over night of:
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI)
•prom+RBS+ctfB+dt (FI) ligated with pSB1c3+prom+RBS+bdhB+dt (FV)

SDS-page of:
•pSB1c3+prom+RBS+ctfA+dt
•pSB1c3+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+bdhB+dt
R:two gels were made for each gene, one was later colored and with the other a western blot was preformed
•we preform western blot for two of the gels and leave the membrane in milk over night in the cold room

2. 9.2015

•we transform the cells with the ligations we set last night
•transformation of BBa_K863010 (lacase)
we set the restriction for and complete the AGE for:
•pSB1c3+bdhB+dt (BI)
•pSB1c3+prom+RBS (BV)
•because we have problems expressing the same constructs we have made before, pSB1c3+GFP (BV) and pSB1c3+ctfB+dt (BI) for our collaboration with Aalto
R: all restrictions were succesful
•we isolate all the restrictions from the gel and set the ligation for 2 hours at room temperature, later transforming the cells pSB1c3+prom+RBS (BV) ligated with bdhB+dt (BI) pSB1c3+GFP (BV) ligated with ctfB+dt (BI)
•we colour the two membranes from yesterday
R: construct with ctfB appears and is partialy in the soluble fraction, the other two construct do not appear for an unknown reason
•we check the expression for both lacases with SDS-page, making two geles, one to be coloured and the other for western blot, storing the membrane in milk over the night in the cold room
R: nothing of note appears on the gels

3. 9.2015

•we colour the membrane from yesterday
R: the membrane does not colour
colony PCR and AGE:
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (back ligation)
•pSB1c3+prom+RBS+ctfB+dt +prom+RBS+bdhB+dt (forward ligation)
R: some samples pSB1c3+prom+RBS+ctfB+dt +prom+RBS+bdhB+dt (back ligation) seem to succed

4. 9.2015

colony PCR and AGE of:
•pSB1c3+prom+RBS+bdhB+dt and •pSB1c3+GFP+ctfB+dt R: the construct with bdhB succeded
•expression of BBa_K733012 and BBa_K863010
R: unsuccesful
•we set restriction and AGE for:
pSB1c3+ctfB+dt as BI and pSB1c3+GFP as BV
R:restriction is succesful
•we isolate both samples from the gel and set ligation over night pSB1c3+GFP (BV) ligated with ctfB+dt (BI)

5. 9.2015

•expression of BBa_K733012 and BBa_K863010
R: unsuccesful
•we set the restriction of pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt as BI, pSB1c3+prom+RBS+ctfB+dt as BI and pSB1c3+prom+RBS+ctfA+dt as BV
R: restriction is succesful
•we isloate restrictions from the gel and set ligation over night
•pSB1c3+prom+RBS+ctfA+dt(BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI) pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•we transform the cels with yesterday's ligation pSB1c3+GFP (BV) ligated with ctfB+dt (BI)
•SDS-page with different samples from yesterday's colony PCR and AGE, we make two gels, one to be coloured, the other for western blot, storing the membrane in milk over night

6. 9.2015

•we colour yesterday's membrane
R: it does not colour
•we transform cells with yesterday's ligations
•pSB1c3+prom+RBS+ctfA+dt(BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI) •colony PCR and AGE of pSB1c3+GFP+ctfB+dt
R: some of the samples are succesful
we set restriction and AGE of:
•pSB1c3+GFP+ctfB+dt as BI
•pSB1c3+prom+RBS as BV
•pSB1c3+prom+RBS+bdhB+dt as BI
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+ prom+RBS+ctfB+dt+prom+RBS+bdhB+dt as BI
R: restrictions are succesful
we isolate the samples from the gel and ligate them over night:
•pSB1c3+prom+RBS (BV) ligated with GFP+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)

7. 9.2015

we transform the cells with yesterday's ligations:
•pSB1c3+prom+RBS (BV) ligated with GFP+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
colony PCR and AGE:
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•pSB1c3+GFP+ctfB+dt
R: some constructs with GFP are succesful
•we set restriction for pSB1c3+prom+RBS+ctfA+dt as BV, do an AGE, isolate the sample from the gel and freeze it

8. 9.2015

•we set restriction for prom+RBS+ctfB+dt+prom+RBS+bdhB+dt as BI, do an AGE, isolate the sample from the gel and set a ligation over night pSB1c3+prom+RBS+ctfA+dt (BV) ligated with pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
We make colony PCR and AGE of:
•pSB1c3+prom+RBS+GFP+ctfB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: some of the samples pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt and pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt are succesful

9. 9.2015

we set restrictions and AGE of:
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt as BI
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+prom+RBS+ctfB+dt as BI
R: restrictions are succeful
we isolate the samples from the gel and set ligation over night:
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with
•prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
SDS-page, we make two gels, one to be coloured, the other for bloting: •pSB1c3+prom+RBS+GFP+ctfB+dt
R: some bands of appropriate size appear
western blot and colouring of pSB1c3+prom+RBS+GFP+ctfB+dt
R: nothing shows on the membrane
we set restrictions over night:
•pSB1c3+GFP as BI
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+prom+RBS+ctfB+dt as BI
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt as BI

10. 9. 2015

we do the AGE of yesterday's restrictions, isolate them from the gel and set ligations for two hours:
•pSB1c3+prom+RBS (BV) ligated with GFP (BI)
•pSB1c3+prom+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
we transform the cells with today's and yesterday's ligations:
•pSB1c3+prom+RBS (BV) ligated with GFP (BI)
•pSB1c3+prom+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt (BI)
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with prom+RBS+ctfB+dt+prom+RBS+bdhB+dt (BI)
•colony PCR and AGE of pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: some samples seem to be succesful

11. 9.2015

SDS-page, we make two gels, one for colouring, other for bloting:
•pSB1c3+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: all samples seem to show the same results
we do western blot on one of the gels and colour the memebrane
R: the membrane shows nothing of note
we set restrictions and AGE, we cut all samples with EcoRl and Pstl to check the size of fragments:
•pSB1c3+prom+RBS+bdhB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: they all cut the same, which means the colony PCR were false pfitives and we have not managed to ligate anything togheter
colony PCR and AGE:
•pSB1c3+prom+RBS+GFP
•pSB1c3+prom+ctfA+dt+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: some samples seem to be succesful
expression and SDS-page of BBa_K733012 and BBa_K863010
R: unsuccesful

12. 9.2015

transformation of BBa_K863005
SDS-page:
•pSB1c3+ctfB+dt
•pSB1c3+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+GFP
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
R: bands appear
we set restriction and AGE:
•pSB1c3+prom+RBS+ctfA+dt as BV
•pSB1c3+prom+RBS+ctfB+dt as BI, BV
•pSB1c3+prom+RBS+bdhB+dt as BI
R: restrictions are succesful
we isolate the samples

13. 9.2015

we set ligation over night:
•pSB1c3+prom+RBS+ctfA+dt (BV) ligated with +prom+RBS+ctfB+dt (BI) pSB1c3+prom+RBS+ctfB+dt (BV) ligated with prom+RBS+bdhB+dt (BI)

14. 9. 2015

•we transform the cells with yesterday's ligations

15. 9.2015

colony PCR and AGE:
•pSB1c3+prom+RBS+ctfA+dt+prom+RBS+ctfB+dt
•pSB1c3+prom+RBS+ctfB+dt+prom+RBS+bdhB+dt
•expression of BBa_K863005

Notebook of testing

23. 3.2015

fermentative production of hydrogen, sampling and analysis on HPLC

24. 3.2015

sampling and analysis on HPLC

25. 3. 2015

sampling and analysis on HPLC

26. 3.2015

sampling and analysis on HPLC

28. 4. 2015

analysis of previous results

2. 5.2015

fermentative production of hydrogen, sampling and analysis on HPLC

3. 5. 2015

sampling and analysis on HPLC

8. 5.2015

learning the basics of working with bacteria, aerobic preparation of 4 different mediums, inoculation, measuring absorbance using spectrophotometry, analysis of previous results

9. 5.2015

learning the basics of working with bacteria, aseptic transfer of bacteria, measuring absorbance using spectrophotometry

10. 5. 2015

learning the basics of working with bacteria, aseptic transfer of bacteria, measuring absorbance using spectrophotometry

11. 5.2015

learning the basics of working with bacteria, aseptic transfer of bacteria, measuring absorbance using spectrophotometry

13. 5. 2015

aseptic transfer of bacteria, aerobic preparation of 4 different mediums, measuring absorbance using spectrophotometry

14. 5. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

21. 5. 2015

learning the basics of working with bacteria, aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

25. 5.2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

26. 5. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

27. 5. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

28. 5.2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

29. 5.2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

3. 6. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

4. 6. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

11. 6. 2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

12. 6.2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

15. 6.2015

aseptic transfer of bacteria into liquid medium, measuring absorbance using spectrophotometry

24. 6. 2015

aerobic and anaerobic preparation of 48 different mediums and saline soluton, aseptic transfer of bacteria onto solid medium plates, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry

25. 6.2015

measuring absorbance using spectrophotometry, analysis of results

26. 6.2015

anaerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

29. 6.2015

aerobic and anaerobic preparation of 72 different mediums and saline soluton, aseptic transfer of bacteria onto solid medium plates, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

30. 6.2015

measuring absorbance using spectrophotometry, analysis of results

1. 7.2015

measuring absorbance using spectrophotometry, analysis of results

2. 7.2015

anaerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

3. 7.2015

measuring absorbance using spectrophotometry, analysis of results

6. 7. 2015

aerobic and anaerobic preparation of 72 different mediums, aseptic transfer of bacteria onto solid medium plates, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

9. 7. 2015

measuring absorbance using spectrophotometry, analysis of results

10. 7. 2015

measuring absorbance using spectrophotometry, analysis of results

13. 7.2015

aerobic and anaerobic preparation of 72 different mediums, aseptic transfer of bacteria onto solid medium plates, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, testing the new Perkin Elmer spectrophotometer, sterilization of working space

14. 7.2015

measuring absorbance using spectrophotometry, testing the new Perkin Elmer spectrophotometer, sterilization of working space, analysis of results

15. 7. 2015

measuring absorbance using spectrophotometry, analysis of results

16. 7.2015

aseptic transfer of bacteria onto solid medium plates, anaerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, sterilization of working space, analysis of results

17. 7. 2015

measuring absorbance using spectrophotometry, analysis of results

20. 7. 2015

aerobic and anaerobic preparation of 72 different mediums, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry

21. 7.2015

analysis of results, ecotoxicological tests, preparing LB agar medium, isolation of n-butanol, measuring absorbance using spectrophotometry

22. 7.2015

analysis of results, ecotoxicological tests, isolation of n-butanol, measuring absorbance using spectrophotometry

23. 7. 2015

analysis of results, ecotoxicological tests, measuring absorbance using spectrophotometry

24. 7. 2015

analysis of results, measuring absorbance using spectrophotometry

3. 8. 2015

aerobic and anaerobic preparation of 72 different mediums and saline soluton, aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

4. 8. 2015

measuring absorbance using spectrophotometry, analysis of results

5. 8.2015

aseptic transfer of bacteria onto solid medium plates, measuring absorbance using spectrophotometry, analysis of results

6. 8. 2015

anaerobic preparation of 72 different mediums, anaerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

7. 8. 2015

measuring absorbance using spectrophotometry, analysis of results

10. 8. 2015

measuring absorbance using spectrophotometry, analysis of results

11. 8. 2015

aseptic transfer of bacteria onto solid medium plates, measuring absorbance using spectrophotometry, analysis of results

19. 8. 2015

aerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry

20. 8.2015

measuring absorbance using spectrophotometry, preparation of Davis minimal medium, preparation of solutions for HPLC and GC methods

21. 8. 2015

measuring absorbance using spectrophotometry, analysis of results, preparation of solutions for HPLC method, measuring the prepared solutions on HPLC and GC

24. 8. 2015

preparation of minimal medium solutions

25. 8.2015

measuring the prepared solutions on HPLC, analysis of results

3. 9. 2015

anaerobic inoculation of bacteria into well plates, measuring absorbance using spectrophotometry, analysis of results

4. 9.2015

measuring absorbance using spectrophotometry

8. 9.2015

measuring absorbance using spectrophotometry, analysis of results

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