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PlastiCure

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When - 30/04/16

Who's In the lab today: Eyal

When - 29/04/16

Who's In the lab today: Ben

When - 27/04/16

Who's In the lab today: Ben

When - 25/04/16

Who's In the lab today: Eyal

	reaction 1	reaction 2	reaction 3	reaction 4	reaction 5	reaction 6	reaction 7
	LCC W.T	LCC C.O	LCC F4	LCC R4	LCC F7	LCC R7	control
pACYC	1.8	1.8	1.8	1.8	1.8	1.8	1.8
Insert	0.9	1.3	0.6	0.8	0.9	0.8	-
Buffer	2	2	2	2	2	2	1.4
DDW	14.3	13.9	14.6	14.4	14.3	14.4	15.2
Ligase	1	1	1	1	1	1	1

incubeted at 16C

M9 salts add to 800 ml H2O:

31.64 gr Na2HPO4*2H20
15 gr Kh2PO4
2.5 gr NaCl
5.0 gr NH4Cl

stirr until dissolved
adjust to 100 ml with distilled H2O

make M9:

700 ml DDW
200ml M9 salts
2 ml 1M MgSO4
100ul 1M CaCl2
adjust to 980 ml with DDW

When - 19/04/16

Who's In the lab today: Efrat

Cyclic Voltametry was preformed in oreder to mesure Cathecol’s redox porential conditions:

2% wt Catechol solution in M9. Potential range:
0-0.5 V.E step: 0.005 V
scan rate: 0.01, 0.005, 0.001 V. Room temperature, pH 7

	0.005 V	0.01 V	0.001 V
E for I max	0.324797	0.334791	0.41574
E for I min	0.119925	0.099937	0.131918
Delta E	0.204872	0.234854	0.283822
E 0	0.222361	0.217364	0.273829

When - 18/04/16

Who's In the lab today: Ben

I loaded the PCR products from yesterday onto an agarose gel (1.5%).

The results:

Lane 1: 1kb plus ladder
Lane 2: pcaG (~660bp)
Lane 3: pcaH (~760bp)
Lane 4: pcaC (~450bp)
Lane 5: pcaD (~840bp)

When - 17/04/16

Who's In the lab today: Ben

I ran a colony-PCR on P. putida with primers for the 4 protocatechuate degredation genes (pcaG/H, pcaC/D)

The reaction mix was:

25μL of ReadyMix Taq PCR reaction mix
22μL of UPW
1.5μL of each primer
A swab from a P. putida colony.

(total reaction volume 50μL)

Cycle parameters were according to Sigma-Aldrich protocol.

ligation of LCC variants into pACYC vector.
Using a ligation calculator in 3:1 ration: we will use about 36 ng of insert for each ligation reaction:

	reaction 1	reaction 2	reaction 3	reaction 4	reaction 5	reaction 6
	LCC W,T	LCC C.O	LCC F4	LCC R4	LCC F7	LCC R7
pACYC	1.4	1.4	1.4	1.4	1.4	1.4
Insert	0.9	1.3	0.6	0.8	0.9	0.8
Buffer	2	2	2	2	2	2
DDW	14.7	14.3	15	14.8	14.7	14.8
Ligase	1	1	1	1	1	1

+control: pACYC 1.4, Buffer 2, DDW 1, Ligase 1
-Incubated overnight at 16C

prepered LB agar plates whith CAM resistance.

When - 16/04/16

Who's In the lab today: Eyal

When - 13/04/16

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

we took Rodococus rubber from glycerol stock and plated on te LB plate at the biological hood.

When - 12/04/16

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

we have made 2 plates containing only LB.
we have made 8 plates containing LB+CRB.

colonies were present.
two- 5 ml starters from CAM plate (pACYC) for midiprep and miniprep.

starter for midiprep 200 ml
midiprep.

When - 11/04/16

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

we took new pACYC from eyal arbely lab, we transformed E. coli it to mg1655 and seed it on CAM plate.

When - 08/04/16

Who's In the lab today: Tomer

we have made 3 medium with glucose, PET, PolyEthylene and palced them in 37C shaker.

When - 06/04/16

Who's In the lab today: Tomer and Ben

restriction of pACYC and E1B GFP

- E1B GFp 1 ul
- 3.1 buffer 5 ul
- XhoI 1ul
- DDW 43 ul
- pACYC 5 ul
- 3.1 5 ul
- XhaI 1 ul
- DDW 39 u
Added 1ul BglII incubeted at 37 C
Glycerol stock made for putida:
- 500 ul from starters
- 500 ul glycerol 30 % +LB

making a startersfor Rodococus rubber:
We took 50 ml of SM+Glucose and added to it some of the glycerol stock.

When - 05/04/16

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

we prepared new LB medium (1L), gathered and prepared glassware for autoclave.
we set up startes for the Putida - for growth experiments and for preparing new glycerol stocks.

We prepared a 20% (w) Catechol solution using 10gr of Catechol (Pyrocatechol from Sigma-Aldrich) and 50ml of DDW.

We prepared M9 Catechol medium using:

10ml M9 Salts
5μL CaCl2 (1M)
100μL MgSO4
39ml DDW
165μL Catechol 20%

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 hour 37 deg
20 min 65 deg
1 ul BalII
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.

	Concentration (ng/uL)	260/280nm
pACYC1	66	1.81
pACYC2	68	1.78

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

LC-cutinase variant	concentration(ng/ul)	260\280
lc-cutinase - WT	43	1.63
lc-cutinase - WT CO	28	1.52
FreeRun4	61	1.71
FreeRun7	47	1.66
ResRun4	41	1.69
ResRun7	46	1.64

WT, WTCO, F4, F7, R4, R7, 1KbPlus led

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

LC-cutinase variant	concentration(ng/ul)	260\280
lc-cutinase - WT CO	134	1.81
FreeRun4	143	1.79
FreeRun7	133	1.80
ResRun4	110	1.78
ResRun7	138	1.81

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

When - 15/03/16

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

We tried constructing a growth curve for E. coli and P. putida on Glucose and Ethylene Glycol.

The Experiment failed (the O.D was declining instead of increasing), probably because the starters we used were kept in the refrigerator for more than 4 days (the bacteria were probably dead).

When - 08/03/16

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

we did a transformation to PACYC, using 50microliter competent bacteria.

100 microliters were used for plating.

an activity test was made of DPNI - we did a transformation in order to check that the plasmid indeed dissolved (nothing is supposed to grow).

concentrations of the genes we received from Weizmann institute:

Lc-cutinase - WT - 26.55 ng/ul
FreeRun4 - 19.15 ng/ul
FreeRun7 - 15.575 ng/ul
ResRun4 - 20.175 ng/ul
ResRun7 - 25.575 ng/ul

When - 07/03/16

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

Starters did not grow.

12 Petri Dishes of LB+CAM were prepared.

When - 06/03/16

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

Preparation of starters for growth curve experiment. (P. putida and E. coli) Glucose and Ethylene Glycol.

When - 25/02/16

Who's In the lab today: Ben

went to the supply room to bring in supplies:

one measuring cup 2L made of glass, and one 1L measuring cup made of plastic.
20ml syringes and 10ml syringes - 10 from each volume. and a few more 2.5 ml because they didn't have 5ml syringes.
around 30 minisart filters.
latex gloves Medium size x5.
A bundle of glass pipettes 10ml (they didn't have 20ml ones) we need to order especially.
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:

Tube	OD (600nm)
Glucose 20%	0.686
Glucose 15% + Ethylene Glycol 5%	0.420
Glucose 10% + EG 10%	0.859
Glucose 5% + EG 15%	0.112
Ethylene Glycol 20%	0.465

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

Tube	OD (600nm)
Glucose 20%	first re-measurement 0.459 second re-measurement 0.611
Glucose 15% + Ethylene Glycol 5%	first re-measurement 0.019 second re-measurement 0.032

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:

Tube	OD (600nm)
Glucose 20%	0.560
Glucose 15% + Ethylene Glycol 5%	0.671
Glucose 10% + EG 10%	0.465
Glucose 5% + EG 15%	0
Ethylene Glycol 20%	0.09

When - 21/02/16

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

how to make competent bacteria?

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.

centrifuge - maximum speed for 15 min and remove the supernatant.
we fill the falcon tube again with glycerol 10%. centrifuge once again for 15 min at max speed and again remove the supernatant.
repeat step 2.
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.
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”.

starting the experiment of growing the Pseudomonas putida on ethylene glycol:

we made the minimal medium where each one has different rates of carbon sources.
we inserted to each medium one colony of Pseudomonas Putida.
we made a blank of 1ml to measure OD.
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

main action - growing two strains of Pseudomonas putida on SOC (EM383, KT2440):

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

main action - making M9 medium for growing Pseudomonas Putida

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.

planning the experiment - we divide the medium into 5 different test tubes where the carbon source will be different in each one:

100% glucose.
75% glucose, 25% ethylene glycol.
50% glucose, 50% ethylene glycol.
75% ethylene glycol, 25% glucose.
100% ethylene glycol.

making the medium:

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

concentrations:

main action - making ampicillin:

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

A protocol for making petri dishes with LB medium:

When - 02/02/16

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

main action - re-sending the two plasmids that got low percentages to sequencing again.

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).

instructions for sending DNA to sequencing:

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

When - 18/01/16

Who's In the lab today: Inbar Segal

Preparation of competent bacteria: E. coli MG1655

Add all of the starter to 500 ml of LB
Use 1 ml of LB as blank, measure OD at time zero at wavelength of 600 nm. (time zero was 0.064 nm)
Measure OD every hour until the bacteria gets to the logarithmic phase (about 3 hours).
Incubar, Shaker, Spectofotometer were used in Ofer yifrach lab (3 floor).
At 11:20 OD was 0.54 nm, we took it out of the shaker and put it in ice.
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

How to make a solution for competent bacteria:

When - 16/01/16

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

Checking the results of the sequencing:

When - 14/01/16

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

How to check the results of sequencing:

Open the file with the graphs displaying the output.
Go to edit->copy seq->FASTA format.
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.
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.
Go to edit->copy seq->FASTA format.
Go to Blast press enter, and paste this part in the same window one row below.
Take the reference and put in a different box/one row below.
Press Blast

Pay attention:

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

Planning a Vector:

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

When - 04/01/16

Who's In the lab today: Dor and Roee

We concerted primers to 10 micro-molars, and sent 11 plasmids to sequencing:

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

Measuring DNA concentration of the plasmids (the numbers match the ones from above):

Plasmid	1	2	3	4	5	6	7	8	9	10	11
concentration (Nano gram/ ul)	222.96	188.87	159.67	125.30	98.86	293.56	153.54	189.91	155.54	157.45	166.29

When - 30/12/15

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

Pcr for two genes (aldA, flco) of the E.coli strain MG1655:

Prepration of a DNA gel

Well	1	3	4	6	7
Sample	ladder	flco	flco control (no template)	aldA control (no template)	aldA

Extraction of plasmids (using the yellow/orange kit):

Number	Plasmid
1	tph A1
2	tctA A1
3	tph A3
4	LC cutinase
5	LC cut + lead seq + his tag
6	tph A2

Number	Plasmid
7	tphB
8	tctA A2
9	rctB B1
10	tph C
11	LC cut + lead seq

When - 29/12/15

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

Making starts for bacteria after Transformation:

Add 5 ml of LB to a 50ml falcon.
Add 5 ul of the matching antibiotic (the ratio is 1:1000).
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.