Difference between revisions of "Team:BGU ISRAEL/Notebook"

When - 23/06/16

Who's In the lab today: Ben and Dar

glycerol stock protocol:

• preparing glycerol:
  • add 10 ml LB to 10 ml glycerol
• preparing the stock:
  1. take 500 ul of starter (with required plasmid transfected)
  2. add 500 ul of the glycerol (from stck)
  3. freeze in -80 C

When - 15/06/16

Who's In the lab today: Ben and Dar

We isolated the plasmids from each cell culture using Geneaid
Again we incubated them with XhoI and XbaI (same protocol as on 14/06)
We ran the samples on a 1% agarose gel with EtBr but using a new loading buffer.

In lanes 6,7,8 we can see good results.
Lane 6 shows the plasmid circular and uncut.
Lane 7 shows the plasmid cut in one location using XbaI.
Lane 8 shows the plasmid circular and uncut (as it does not have a XhoI restriction site).

starters growth for activity testing of LCC:
LB 50 ml+ CP 50 ul.
each tube will get 5 ml.
In addition, LB 5ml without antibiotic for BL21 growth without insertion.
For each tube. we added colony with another varient+2 control tube.
the colony that we are testing:
pACYC LCC WT C6
pACYC LCC C.O C4
pACYC LCC F4 C2
pACYC LCC F7 C3
pACYC LCC R4 C7
pACYC LCC R7 C2
pACYC no insert
BL21 (without antibiotic)

Turbidity check and induction:
For LB 7 ml + CP 7ul we added 70 ul for each starter. ( 37C 200 rpm)

PET degradation test:
we cheked if there is any change in PET wight after one day:

When - 14/06/16

Who's In the lab today: Ben and Dar

We isolated the plasmids from each cell culture using Geneaid Presto mini plasmid kit.
We Incubated each of the plasmids with XhoI and XbaI restriction enzymes (2 hours incubation in 37 degrees and 20 minutes in 65 degrees).
We ran the results in a 1% agarose gel with EtBr. Both plasmids show on the gel uncut (lanes 2-3). We have no ability to determine their size (as they are circular).
We put new starters for another preparation of plasmid DNA.

When - 13/06/16

Who's In the lab today: Ben and Dar

We incubated starters for the pSEVA224 and pSEVA414 in 30 degrees.

When - 11/06/16

Who's In the lab today: Eyal and inbar b

compentent cells prepretion

When - 10/06/16

Who's In the lab today: Eyal

preparation of TRIS-HCL:

1. for prvpB degredation---> C1V1=C2V2→ 1M*V1= 25mM*9.9ml→ V1=247.5 ul, DDW=9.6525ml
2. for PET degredation---> C1V1=C2V2→ 1M*V1= 500mM*2ml→ V1=1ml, DDW=1ml

preparation of pNP-B:

• pNPB - 35ul -----------> 1 ml of pNPB ---------> 100 mM into
• acetonitrite - 965 ul --------> in 200 mM conc. ----->9.9 TRIS 25 nM

we left 8 tubes with LCC variants for incubation at 10:30 AM for the weekend (50 C)

When - 08/06/16

Who's In the lab today: Eyal

When - 06/06/16

Who's In the lab today: Eyal

we designed an expriment to test differnet carbon surce from our metabolic pathway for putida.
We used 96DW and we orgenized it as seen in the picture:

this experiment will last for one week we will check the change in the turbidity with plate-reader.

When - 31/05/16

Who's In the lab today: Eyal

we cut De-lorenzo plasmids (414+224), we run thus plasmids on agar gel for analazing if thus plasmids were cut.

When - 28/05/16

Who's In the lab today: Eyal

starters for Putida KT2440.
5 ml LB+5 ul amp, 100ng/ul, 30C

transformetion for pACYC plasmid without insertion(negative control for coplement cells) and with insertion LCC WT C6, LCC CO C4, LCC F4C2, LCCF7C3, LCC R4C7, LCC R7C2 to BL21 bacteria for protein exprection.

• 8 tubes with compatent BL21 bacteria, 80C,10 min.
• 1 ul from each plasmid for each tube.
• mixed with tip.
• keep in ice for 20 min.
• keep in ice for 3 min more.
• 500 ul SOC without antbiotic.
• 27C 1h
• centrifugation 5000 rpm 2min
• take off 500ul from the liquid.
• mix the residue with the rest liquid.
• sowing in LB+CMP

When - 24/05/16

Who's In the lab today: Eyal

seq of LCC variants 3 ul primer in each reaction 300 ng DNA.
(each with Rw\Fw primer).

When - 19/05/16

Who's In the lab today: Roee and Neomi

We made plates with : LB+TET, LB+Strep. we took the starters with De-Lorenzo bacteria to swo on thus plates.

After all the starters and glycerol stocks were found contaminated a new plate of rhodococcus ruber was recieved from alex sivans lab. a new SM medium was made and we have set the tools inorder to grow new starters on sunday may the 22nd.

When - 13/05/16

Who's In the lab today: Eyal

Constructing a growth-curve for the Putida on Cathecol. The experiment was contaminated. we will have to repete it.

When - 11/05/16

Who's In the lab today: Eyal

pSEVA starters from 10.5, and sowing on LB+KM 2 ml starters 300 rpm, 2 min, RT, using sup and sowing the output.

When - 03/05/16

Who's In the lab today: Eyal, Roee and Dor

one colony from each plate was sent fot seq(because of the bw concentration, 15 ul from each stock was sent)

2 starters of rhodococcus ruber were made from two different glycerol stocks to check for contamination. each sample was plated on a nutrient agar plate that was divided in to two parts one for each glycerol stock. the starters were put in ramons lab in the incubator shaker 37 degrees and the plate was put in our incubator at 37 degrees as well.

we prepered glycerol stocks of BL21.

When - 31/03/16

Who's In the lab today: Dar

checking whether the Putida from the experiment last week (Growth curves for Putida and E.Coli on EG) are still alive (and that the OD we’ve measured isn’t the result of dead bacteria).
at both times samples were taken from the liquid solution in the tub and put on plates (LB+amp). at both times growth on the plates was observed - the bacteria were still alive.

When - 28/03/16

Who's In the lab today: Inbar S and Eyal

Try #3 of the pACYC restriction
This time - 1 hour with each enzyme and run on agarose gel 2ul after each cut.

• pACYC 2000ng 30ul(66ng/ul)
• DDW 58ul
• 3.1 Buffer 10ul
• XhoI 1ul

1. 1 hour 37 deg
2. 20 min 65 deg
3. 1 ul BalII
4. 1hour -> run on agarose gel 2ul.

When - 28/03/16

Who's In the lab today: Inbar S and Eyal

Another restriction reaction for the pACYC plasmid.
We used the pACYC2 product from 25/03(68ng/ul) - Restriction requires 2000ng of plasmid DNA so we used a volume 29.4ul.

Reaction protocol:

• DNA 29.4ul
• 3.1 Buffer (NEB) 10ul
• BalII 1ul
• XhoI 1ul
• DDW 58.8ul

No Bands again.

When - 27/03/16

Who's In the lab today: Dar

Setting up the growth-curve experiment for both the Putida and E.Coli (MG1655) on Ethylene Glycol.
This time the experiment will take a week - 2-3 measurements per day.

When - 25/03/16

Who's In the lab today: Inbar

Mini-prep for pACYC.

When - 24/03/16

Who's In the lab today: Tomer and Inbar Bariah

Removing the putida starter from the incubator to the fridge.

DNA extraction from reaction mix- LC-cutinase variants.
total volume 30ul

In addition, starters for pACYC were prepared:
10ml LB+10uL CAM for 2 starters.

When - 23/03/16

Who's In the lab today: Inbar Segal, Dar, Ben, Inbar B, Roee, Dor, Tomer and Eyal

Produced the pACYC plasmid from the starter and cleaned it using mini prep kit.
Concentration: 52ng/ul 260\280: 1.83

another try at constructing a growth-curve for the Putida and E.coli on Ethylene glycol and Glucose.

Setting up new starters for the Putida and E.coli (MG1655)

When - 22/03/16

Who's In the lab today: Eyal and Inbar Segal

Cutting LC-cutinase and pACYC vector with restriction enzymes, no bands were visible on electrophoresis gel.

When - 18/03/16

PCR extraction of LC-cutinase variants
total volume 30ul

When - 17/03/16

Who's In the lab today: Inbar Bariah

PCR to amplify LC-cutinase variants: WT CO, FreeRun4, FreeRun7, ResRun4, ResRun7.
WTCO, WTCOX,F4,F4X,F7,F7X,R4,R4X,R7,R7X,1KbPlus led

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When - 25/02/16

Who's In the lab today: Ben

1. one measuring cup 2L made of glass, and one 1L measuring cup made of plastic.
2. 20ml syringes and 10ml syringes - 10 from each volume. and a few more 2.5 ml because they didn't have 5ml syringes.
3. around 30 minisart filters.
4. latex gloves Medium size x5.
5. A bundle of glass pipettes 10ml (they didn't have 20ml ones) we need to order especially.
6. big tank for water (needs a wash and then we can use it for ddw or upw).

When - 24/02/16

Who's In the lab today: Neomi, Tomer and Dor

a glycerol stock of rhodoccocus ruber was prepared from the starter made yesterday. I didn't know if the glycerol in our fridge was autoclaved so I borrowed from Ramon's lab, the stock is at (-80) degrees Celsius at Ramon's shelf.

Removed the putida from the incubator after growing on ethylene glycol and glucose and checked the OD (600nm)
Results:

because the measurements were inconsistent I made several measurements. especially tubes 1 and 5:

When - 23/02/16

Who's In the lab today: Ben and Roee

• a starter of Rhodococcus ruber was made. it included 50ml SM(medium) + glucose + microelements. the work was done in a biological hood afterwards it was placed in a shaker at 37 degrees Celsius overnight.
• taking the putida out of the shaker after growing it on glucose and ethylene glycol and checking OD (600nm).
  Results:

When - 21/02/16

Who's In the lab today: Roee, Dar, Dor and Neomi

• all the process need to occur on ice, in a centrifuge set to 4degree Celsius, and the glycerol need to be cold.
• first of all, we grow the bacteria over night with antibiotics -> in 50 ml falcon tube.

1. centrifuge - maximum speed for 15 min and remove the supernatant.
2. we fill the falcon tube again with glycerol 10%. centrifuge once again for 15 min at max speed and again remove the supernatant.
3. repeat step 2.
4. we combine both tubes (if we have more than one, if not we wash it again with glycerol as we did in 2+3) after we fluidize the bacteria in glycerol we complete the volume with 10% glycerol, we centrifuge and remove the supernatant.
5. fluidize the sediment with 2 ml glycerol and divide into tubes 50 microliter in each one and freeze with liquid nitrogen.

a medium for growing Rhodococcus Ruber was made in the solution are missing microelements that we are supposed to receive from Valentina and there is no carbon source. the solution is in the fridge door with a sticker “rhodococus ruber solution”.

there was no CaCl2 2H2O si we used 680microliter of CaCl2 1M from the fridge.

When - 22/02/16

Who's In the lab today: Roee, Dar, Dor, Tomer, Inbar Segal and Eyal

competent bacteria were made from the putida KT2440 strain. the bacteria were divided into 25 Eppendorf's with 50microliter each (the volume needed for each transformation) the Eppendorf's are in the freezer (-80 degrees) of Ramons lab in a red box the with the inscription “Ashalim”.

1. we made the minimal medium where each one has different rates of carbon sources.
2. we inserted to each medium one colony of Pseudomonas Putida.
3. we made a blank of 1ml to measure OD.
4. we put the bacteria in a Shaker set to 37 degrees Celsius in Naama's lab.

We did a PCR reaction for the LC-Cutinase + leader Sequence Gene. in order to take it from the iGEM plasmid and insert it into a PACYC vector. we ran the PCR products on an agarose gel using electrophoresis and took a picture. we got one big band in the size we wanted 1KBP~ and a second band, weaker one in the size of the entire plasmid.

When - 15/02/16

Who's In the lab today: Inbar Bariah

inserting a Q-tip that touched the glycerol stock of the relevant culture into 3ml of SOC with ampicillin (needs to be near fire) and growing overnight. -results- the bacteria grew and the medium became very murky. the bacteria were afterwards grown on petri dishes with LB + amp from every strain we inserted 20 microliters and we also made plates where we isolated via striking.

colonies from every strain (KT2440, EM383) were moved into a liquid LB medium for incubation overnight in 37 degrees Celsius.

When - 14/02/16

Who's In the lab today: Inbar Bariah, Neomi and Tomer

Instead of glucose as a carbon source we planned an experiment where the ratios between glucose and ethylene glycol vary in order to check if the bacteria can use ethylene glycol as a carbon source. if so, we won't need to insert the two plasmids we took from E. coli.

1. 100% glucose.
2. 75% glucose, 25% ethylene glycol.
3. 50% glucose, 50% ethylene glycol.
4. 75% ethylene glycol, 25% glucose.
5. 100% ethylene glycol.

1. with the assistance of Idit we made MgSo4 and a Salt sterile solutions using autoclave.
2. we used Sterile CaCl that already gone through autoclave and also DDW that we already had in the lab.
3. we made 20% glucose and filtered it.
4. we filtered ethylene glycol and made a 20% ethylene glycol solution.

concentrations:

1. weighing 1g ampicillin.
2. adding 10ml DDW.
3. filtering with syringe 0.24 wide.
4. dividing it into 20 Eppendorf's (500microliter each).

When - 02/02/16

Who's In the lab today: Roee, Inbar Bariah and Tomer

after checking once again the sequences in blast of the two plasmids that got low percentage and after consulting with Dr.Birnbaum and Naama we understood that the way we looked at the percentage wasn't correct and that the sequencing was actually correct and we had the sequence we wanted on the plasmid. (before we knew about the sequencing being correct we made Chloramphenicol in working dosage in order to grow the bacteria that carried the plasmid on the medium we wanted).

1. Plasmid Dosage need to be between 75-100 ng/ul.
2. volume needed: ten microliters per plasmid.
3. Primers - 2 microliters per sequenced plasmid (minimum volume 5 ul).

When - 18/01/16

Who's In the lab today: Inbar Segal

1. Add all of the starter to 500 ml of LB
2. Use 1 ml of LB as blank, measure OD at time zero at wavelength of 600 nm. (time zero was 0.064 nm)
3. Measure OD every hour until the bacteria gets to the logarithmic phase (about 3 hours).
4. Incubar, Shaker, Spectofotometer were used in Ofer yifrach lab (3 floor).
5. At 11:20 OD was 0.54 nm, we took it out of the shaker and put it in ice.
6. We checked if the bacteria can grow on agar with kan and amp - we did not see any colony.

When - 17/01/17

Who's In the lab today: Dor, Inbar Barih and Neomi

When - 16/01/16

Who's In the lab today: Dor, Roee and Tomer

When - 14/01/16

Who's In the lab today: Dor, Roee, Tomer and Naama

1. Open the file with the graphs displaying the output.
2. Go to edit->copy seq->FASTA format.
3. Go to Blast and paste what we copied in the first window, if we copied first the sequence of the foreword primer after we will do for the reverse primer and vice-versa.
4. Open the file with the graphs displaying the output, but this time for the reverse primer if we opened its first for the foreword and vice-versa.
5. Go to edit->copy seq->FASTA format.
6. Go to Blast press enter, and paste this part in the same window one row below.
7. Take the reference and put in a different box/one row below.
8. Press Blast

1. In the top of the page in Blast you can check if R or F fits the reference.
2. It's better to check if R and F match (we expect it to be similar not the same).
3. Notice the values of query-how similar are the samples, ident-percentage of identical bases.
4. Look where most of the different bases are located (hopefully in the end or the beginning).
5. Small letters instead of big ones (atg and not ATG) marks that this sequence repeats itself.
6. If you to write comments, write according to the locations of the reference.

1. Look at the map of the vector that was sent (for the Putida) try to see which restriction enzymes are relevant.
2. Go to the website-nebcutter, check for each of the fragments which restriction enzymes are relevant (each fragment separately, according to the reference).
3. press custom digest for the list of enzymes, check that this enzyme don't cut inside of the fragments.
4. choose restriction enzymes.
5. Plan primers with tails that will match the enzymes.
6. pcr and ligation.
7. transformation.

When - 04/01/16

Who's In the lab today: Dor and Roee

• LLK - lc cutinase + led seq + his tag
• LL - lc cutinase + led seq

When - 31/12/15

Who's In the lab today: Liran and Tomer

When - 30/12/15

Who's In the lab today: Ben, Liran, Dar, Tomer and Dor

1. Prepration of a DNA gel

When - 29/12/15

Who's In the lab today: Inbar Barih, Inbar Segal, Eyal, Liran and Ben

1. Add 5 ml of LB to a 50ml falcon.
2. Add 5 ul of the matching antibiotic (the ratio is 1:1000).
3. With a toothpick stab one colony spread it on another Agar plate (in case the starter wont work we will be able to use that colony again) and then throw it inside of the 50 ml flacon.