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From 2008.igem.org

July 22nd

We have 4 tubes from last year. These strains are frozen in glycerol.

• PNZ8901 plasmid in E.coli MC1061 Strain
  • Chloroamphenicol resistant
  • "sure" vector
  • does not integrate

• B. subtilis strain 1A1
  • No resistance
  • Same as strain 168 but tryptophan deficient

• E.coli strain MC1061, no plasmid

• PP182 plasmid in E.coli strain DH5alpha
  • Ampicillin resistance
  • integrates

Checking antibiotic resistance

Purpose : Grow each of them on a plate to test their antibiotic resistance

Protocol

• Warm up frozen tubes
• Take 15uL of each and put it on plates without antibiotic
• Incubate at 37°C

July 23rd

• Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics

1/ Purpose Check antibiotic resistance of our different strains

Strain	Plasmid	Extracted from	Antibiotic use	Concentration of antibiotic
DH5alpha	PP182	tube	Amp	100
MC1061	NONE	tube	Cm	35
MC1061	PNZ8901	tube	Cm	35
DH5alpha	PP182	plate	Amp	100
MC1061	NONE	plate	Cm	35
MC1061	PNZ8901	plate	Cm	35

2/ Purpose : Find the right concentration of antibiotic so that B. subtilis survive

- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB

- Incubate at 37°C

3/ Purpose : Grow plasmids in TOP10, transformation

4 plasmids :

• I746000
• I746100
• I746101
• I746001

1 control : PUC19

- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf

- 30min on ice

- Heat shock : 60s at 42°C

- 2min on ice

- Add 89.5uL of SOC

- 60min at 37°C

- Put the mix on plate with ampicillin resistance

- Incubate at 37°C

July 24th

• Test of antibiotic resistances of strains of last year

Strain	Plasmid	Extracted from	Antibiotic use	Observations	Conclusion
DH5alpha	PP182	tube	Amp	Many colonies	Amp Resistance OK
DH5alpha	PP182	plate	Amp	Many colonies	Idem
MC1061	NONE	tube	Cm	Nothing	As expecting, no Cm Resist.
MC1061	NONE	plate	Cm	Nothing	Idem
MC1061	PNZ8901	plate	Cm	Maybe a few colonies	Contamination??
MC1061	PNZ8901	tube	Cm	No colonies	no Cm Resist.

• Transformation of E.coli with different plasmids from last year

Strain	Inserted plasmid	Antibiotic	Observation	Conclusion
E.coli	I746000	Amp	No colonies	Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli	I746100	Amp	No colonies	Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli	I746001	Amp	Many colonies	Transformation OK
E.coli	I746101	Amp	Many colonies	Transformation OK

Antibiotic test for Bacillus resistance

We want to find the lowest concentration of antibiotic which kills Bacillus.

Dilution of Amp. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL)	100	75	50	25	10
100 mg/mL Stock	1:1000	3:4000	1:2000	1:4000	1:10000

Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL)	35	25	15	10	5
100 mg/mL Stock	1:1000	1:1400	3:7000	1:3500	1:7000

• Add Disks in antibiotic solution for 5 mins [ Protocol to sterilize tweezers : Wipe with Kimwipes, Ethanol, Flame ]
• Melt Soft Agar
• 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08
• Pour SA+Cells over blank hard agar plates
• put disks with different concentration of Amp/Cm above SA
• Incubate at 37°C

Results [Plates were prepared before lunch, at 5pm there were visible growth]

-Amp Plates: Huge rings of no growth around 100, 75, 50, 25, and 10.

-Cm PLates: Tiny rings of no growth around 5, 10, 15 and 25. Small ring of no growth around 35 but not well defined. (No clear zones)

Salts for making Bacillus Competent

10x Medium A Base

• Yeast Extract 10g
• Casamino Acid 2g
• add Distilled water to 900mL

-Aliquot into 5 different bottles. [180mL each]

10x Bacillus Salts

• NH4)2SO4 20g
• K2PO4 anhydrous 139.66g
• KH2PO4 60g
• Tri-Sodium Citrate 10g
• MgSO4.7H2O 2g
• Add Distilled water to 1000mL

-Aliguot into 5 different bottles. [200mL each]

Medium B

• • Preparing 50mM CaCl2.2H2O
    • CaCl2.2H2O 1.470g
    • Add Distilled water to 20mL [Final conc. 500mM]
    • Take 10mL of 500mM
    • Add 90mL of distilled water [Final conc. 50mM]

• • Preparing 250nM MgCl2.6H2O
    • MgCl2.6H2O 0.508g
    • Add Distilled water to 10mL [Final conc. 250mM]
    • Take 1mL of 250mM
    • Add 99mL of Distilled water [Final conc. 250μM]
    • Take 1mL of 250μM
    • Add 99mL of Distilled water [Final conc. 250nM]

• Add 100mL of 50mM CaCl2.2H2O
• Add 100mL of 250nM MgCl2.6H2O

-Total volume 200mL

NOTE: To complete Medium B, Take 0.2mL of this solution

July 25th

Results from Yesterday

• Antibiotic test

Results for Amp

Even with a concentration of 10μg/mL, Amp kills Bacillus. Since we want to know which is the lowest concentration of Amp which kills B.S, we are going to test with some lower concentrations.

Results for Cm

Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.

Wet Work

• New antibiotic tests

Dilution of Amp

Concentration (μg/mL)	10	7.5	5	2.5	1
10 μg/mL Stock (from yesterday)	1:1	3:4	1:2	1:4	1:10

Dilution of Cm

Concentration (μg/mL)	35	37.5	40	42.5	45
35 μg/mL Stock (from yesterday)	1:1	5:6 (from 45μg/mL)	1:875	17:18 (from 45μg/mL)	9:7000

- Melt Soft Agar - 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08 - Pour SA+Cells over blank hard agar plates - Put 5 blank disks on each agar plate - Add 40μL of antibiotic on each disk - Incubate at 37°C

Results

Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!

July 28th

• Results of antibiotic plates from yesterday

For Cm, 35μg/mL should be enough to kill B.S.

For Amp, nothing can be concluded!

Wet Work

• Check plasmid ppL82

We had 2 samples of ppL82 in DH5α in LB solution (~4mL), one from the frozen glycerol tube and one from a colony picked on a plate. Normally plasmid and cells should not be kept in freezer. So, we want to extract this plasmid, check its size and then keep it in the fridge. We do that for the 2 different samples.

- Plasmid Miniprep (standard protocol)

- Test the concentration of DNA in each tube

1. ppL82 plate : 89.8ng/mL
2. ppL82 tube : 71ng/mL

• Preparation of different stocks of strains and plasmids

PNZ8901

- Plate a single colony (from 23/07/08) on a Cm plate

- Incubate at 37°C

- Grow the entire frozen glycerol tube in 20mL of LB without antibiotic to check if this stock is still good (we will check the plasmid on a gel tomorrow)

- Incubate at 37°C

MC1061

-Pick e single colony of MC1061 (from 22/07/08) and put it in 10mL of LB (to make glycerol stocks)

-Incubate at 37°C

1A1

- Add 100μL of LB+1A (mix of friday) and 10mL of LB

- Incubate at 37°C

• Make B.S. 1A1 competent and transform them

- Prepare Medium A

Add 81mL of SDW, 10mL of 10X Medium A base and 9mL of 10X Bacillus salts

- Make B.S. 1A1 competent

1. In 10mL of medium A and add about 10 colonies of B.S.
2. Check the OD650, you should have an OD between 0.1 and 0.2. t0 for OD = 0.1876
3. Check the OD650 every 20min and plot OD against time on semi-log paper. When the point at the culture leaves log growth, it is ok

Time (min)	0	20	40	60	70	80
OD650	0.1876	0.2074	0.3282	0.4545	0.4895	0.5040

At, t0 = 70min, log growth seems to stop.

1. Incubate at 37°C 90min after t0
2. Warn 10 Ependorf tubes with 0.45mL of Medium B
3. Add 50μL of culture in each tube of Medium B at t90
4. Incubate the diluted culture at 37°C with vigorous aeration for 90min

- Storage : we want to store 7 tubes

6 tubes in the freezer (with 60μL of glycerol)

1 tube on the bench

- Transformation

1. We make ppL82 from plate, ppL82 from tube, and a control
2. Add 0.5μg of DNA (15μL)
3. Incubate at 37°C for 30min
4. Plate on Cm plates

July 29th

• Result from the gel (29/07/2008)

• Lane8 : ppL82 (plate)
• Lane9 : ppL82 (tube)
• Lane 10 : I746001
• Lane 11 : I746101
• Lane 12 : hyperladderI

The ladder seems to be wrong. So it was really difficult to ckeck the size of our plasmid ppL82. To make, that, we assume that the size of our biobricks was ok, and we estimate the size of our plasmid. The plasmid ppL82 seems to have the right size, but we will have to check again to be able to use a ladder.

• Competence of B.S. kept on the bench

We observed B.S. with a microscope. Problem, all B.S. seems to be dead!

Possible reasons of this problem :

- Problem with the vector (it could be a wrong vector, we are not really sure)

- Quantity of DNA, we added 0.5μg. The protocol was 1μg, but normally it should be ok.

- B.S. may not be competent (we have to test motility with microscope)

- Cells could be dead : replate them

- Try to add tryptophan in the medium

Probable reason : we forgot to dilute the medium B, that's why it did not work

• Results from transformation plates (B.S.)

There are 2 colonies on a plate, but it does not look like Bacillus. Moreover, there is also a colony on our negative control plate. SO it must be contamination (resistant to CM!). We will do the transformation again.

Wet Work

• Prepare medium for transformation of B.S.

- Preparation of medium B: 10mL of medium A and 0.2mL of 50mMCaCl22(H2O) + 250mMgCl26(H2O)

- Preparation of medium A with tryptophan : 81mL of SDW, 9mL of 10X Bacillus salts, 10mL of 10X Medium A base and 0.1mL of Tryptophan (11mg/mL)

• Check plasmid PNZ8901

- Plasmid miniprep (same protocol with 60μL of elution buffer)

- Digest : with PstI and SalI from Biolabs, and Buffer 3 (add 15μL of DNA)

- Gel (17μL of sample and 3μL of dye)

Results from the gel

• Lane9 : PNZ8901
• Lane10 : HyperladderI

The sizes we expected were about 1100kb and 2100kb. The sizes should be ok!

July 30th

• Transforming Bacillus Subtilis with medium A and medium A + tryptophan

Medium A

Time (min)	0	20	40	60	73	85	95	105
OD650	0.1394	0.1370	0.1660	0.2304	0.2778	0.3138	0.3373	0.3583

For this medium, the curb stop to increase logarithmically at 100min.

Medium A + tryptophan

Time (min)	0	20	40	60	73	85	95
OD650	0.1493	0.1636	0.2080	0.2909	0.3512	0.4002	0.4165

For this medium, the curb stop to increase logarithmically at 90min.

- Incubate 90min at 37°C by shaking

- Prepare 7 tubes for medium A and 7 tubes for medium A + tryptophan : add 0.45mL of prewarmed medium B and 0.05mL of culture and incubate 90min at 37°C by shaking.

- Transform B.S. by adding DNA and incubate 30min at 37°C

We have one transformation with ppL82 (add 7.5μL of DNA), one with PNZ8901 (add 10μL of DNA) and one negative control without DNA.

- Plate 500μL on Cm plate (35μg/mL)

- For the remaining tubes, in one, add 60μL of glycerol and keep it in the freezer and keep the other one on the bench (for each medium)

• Checking our big stock of biobricks and PNZ plasmid

We did big culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.

- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks

- Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation

	260/280	DNA concentration (ng/nl)
I746001	1.79	50.5
I746101	1.87	54.3
PNZ8901	1.84	87

- Digest

For I746001 and I746101	For PNZ8901 plasmid
14μL of SDW	14μL of SDW
2μL of 10X Fast Digest Buffer	2μL of Buffer 3 (Biolabs)
2μL of DNA of biobricks	2μL of DNA of PNZ8901
1μL of EcoRI	1μL of PstI
1μL of SpeI	1μL of SalI

- Gel

• Results from this gel

• Lane 9 : I746001
• Lane 10 : I746101
• Lane 11 : PNZ8901
• Lane 12 : HyperladderI

Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.

• New gel to check

- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901).

- Do single digest for PNZ8901 (one with PstI, one with SalI)

- Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)

• Result from this second gel

The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem. With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks.

Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.

August 1st

• Grow received vectors

Vectors	AmpR (μg/μL)	CmR (μg/μL)	KanR (μg/μL)
ECE112	100	0	0
ECE147	50	0	0
ECE149	50	0	0
ECE150	50	0	0
ECE151	50	0	0
ECE153	100	0	0
ECE162	100	0	0
ECE165	100	10	0
ECE166	100	10	0
ECE171	50	0	10
ECE172	100	10	0
ECE176	100	5	0

- Preparation of Agar plates

We have bottles of 200mL.

Type	Amp (μL)	Cm (μL)	Kan (μL)
Amp100	200	0	0
Amp50	100	0	0
Amp100 + Cm10	200	57.1	0
Amp100 + Cm5	200	28.6	0
Amp50 + Kan10	50	0	80

- Preparation of LB tubes

We prepare 10mL of LB in which we add a single colony.

Type	Amp (μL)	Cm (μL)	Kan (μL)
Amp100	10	0	0
Amp50	5	0	0
Amp100 + Cm10	10	2.9	0
Amp100 + Cm5	10	1.4	0
Amp50 + Kan10	5	0	4

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