Our Protocols

Lab Book group 3"The subtile ones"

Induced Sporulation according to protocol Sporulation
5 samples were inoculated to grow over night
diluted them to an OD= 0.1 (around), incubate them till an OD of 0.8 (so that they are in the exponential growth phase, probably takes around 3 hours)
Put an equal amount of DSM Culture medium and the sample in a reaction tube and were inoculated for 24 hours to from spores
10 Sporulations with 0,5ml Bacillus and 0,5ml DSM were induced

5.07.2016

(DS)

Strains B52-B56 were inoculated in 5 mL LB-medium to grow over night

6.07.2016

Germination

(DS)
sample of spores were placed in LB-media to germinate according to protocol Germination + Spore Number
Grown B52-B56 were put in 5 ml Cm (1 to 1000 dilution) and let them grow over night
–> was a too high amount of Cm on the plates so most of the cultures did not survive (only surviving bacteria from strain B55)

7.07.2016

(DS)
Grown samples B52-56 were put in 5ml + Cm (1 to 5000 dilution) and let them grow over night to test their resistance against Cm
Induced Sporulation with the B55 colony (2 samples with 9ml, let them grow till an OD of 0.8)–>contaminated

8.07.2016

(DS)

Glycerol stocks (B52-56)

Let the GFP-displaying-strains colonies (B52-56) grow in 9 ml LB+Cml (diluted 1 to 5000)
Sporulation of the GFP-displaying-Bacillus: 5 samples in 5ml DSM (5 samples with 1ml bacteria(centrifuged and wash with ddH2O) and in 0,5ml DSM)let it sporulate for 24hours
Glycerol stocks : B52-55 (500ul culture + 500ul Glycerol 30% in an eppi and then in the freezer -80°C)
GFP-displaying-Bacillus were inoculated (5ml LB + 10 ul Cml)
WT 168 inoculated (6,5mL LB)

9.07.2016

(DS&YS)

Sporulation

Induced sporulation of the GFP-displaying-Bacillus (5 samples with 1ml bacteria(centrifuged and wash with ddH2O) and 0,5ml DSM)
Picked a colony from the WT 168 plate and put them into 2ml LB (for a glycerol stock tomorrow)
GFP-displaying-Bacillus (B52-56) were inoculated (5ml LB + 10 ul Cml)
Germination from Spores from WT (DSM10) successful

10.07.2016

(DS)

Glycderol stock (WT168)

Germination

Glycerstock was created from WT168
Strains B44& B49 were put into 5LB to grow over night
Started to work on competent cells (create competent cells from B52-56)

Dilute the stains on a plate with Cml (7ul Cml + 1ml LB on a normal LB plate)

Induced germination with the germination-knock-out-strains(B46 & B 47) in 5LB (dilute 10ml LB with 2ul Kan + 10ul Cml + 20ul spec)→ did not work
Induced germination with the WT168 (20ml LB) and B53 (20ml LB+ cml)

11.07.2016

(DS)

new plates

plated the B44 (cotZ) & B49 (cgeA) from munich (Julia Bartels)
Germination knock-outs (B46 and B47) were inoculated in 5 ml LB to grow over night

12.07.2016

(DS;YS)

Prepare medium for competent cells

Selected single colonies from B44 and B49 to grow over night
Germination knock-outs do not grow –> were already spores (germination knock-out works)
Prepared Bacillus Salts and 100ml Medium A base according to Making competent B subtilis. Incubated B44,B49 and WT168 single colonies over night in Medium A (to make them competent)
Inoculated B35 in 3ml (Lb+Cml) over night

13.07.2016

(DS;YS)
Making the cells competent did not work, there was no visible growth
Plated the B46 and B47 colonies (Germination K.O.)

14.07.2016

(DS,YS)

no growth in medium A

Tried to make competent cells again –> there was no growth of B.subtilis
Get a single colony from WT168 for the colony PCR and let them grow over night
Picked a single colony from B46 and B47 to incubate over night (to induce sporulation and check for germination)

15.07.2016

Sporulation

Sporulatin was induced with the strains WT, B47, B46
dilute them to an OD of around 0,1 and let them grow till they reach their exponential growth phase (around OD 0,8 = aroud 2 and 1/2 hours)
they were wash once in ddH2o and put in DSM medium to grow for 24 hours

18.07.2016

(DS)
inoculated B52-56, WT 168 and B46 in 5ml LB over night in the incubator

19.07.2016

(DS)
sporulation was induced of before mentioned samples according to protocol Sporulation

20.07.2016

(DS)

Cell counting Neubauer counting chamber

cells were counted with a Neubauer-chamber with a dilution of 1 to 10
Spore number:
B56 = 317440000 spores/ml
B55 = 281600000 spores/ml
B54 = 312320000 spores/ml
B53 = 235520000 spores/ml
B52 = 302080000 spores/ml
B46 = 343040000 spores/ml
WT168 = 348160000 spores/ml

Aliquots of spores

samples were diluted with DSM (2ml bacteria with 2ml DSM) so that the spores are around 100 million spores/mL
aliquot (6x400ul per sample) + freezing the samples

spore number in 400ul:
B56 = 63488000 spore/400ul
B55 = 56320000 spore/400ul
B54 = 62464000 spore/400ul
B53 = 47104000 spore/400ul
B52 = 60416000 spore/400ul
B46 = 68608000 spore/400ul
WT168 = 69632000 spore/400ul

21.07.2016

(DS;YS)
Medium for competent cells + DSM starter medium created
B52 and B53 in 3ml LB +Cml and WT168 in 4 ml LB were inoculated to grow over night in the incubator

22.07.2016

(DS)

Spores heat shock and sonification (not successful)

Sporulation was induced with the strains B52, B53 and WT168 in 5ml DSM
picked single colonies from WT168; B46; B47; B44 & B49 to grow over night in 5 ml LB
Spores B46 and B52 were sonicated. 400ul of the colonies were put in an eppi, zentrifuged and the resting pellets were washed with 1% PBS buffer, centrifuged again and sonicated with 500ul PBS buffer
Spores from B46 (lysed and none lysed) and B52 (lysed and none lysed) were put in 5ml LB to determinate their growth

23.07.2016

(DS)
Spores WT168 and WT168 were sonicated. 400ul of the colonies were put in an eppi, zentrifuged and the resting pettet was washed with 1% PBS buffer, zentrifuged again and sonicated with 500ul PBS buffer
the sonicated cells were plated to see their growth –> both plates show growth , so the sonication did not work

24.07.2016

(DS)
Sporulation was induced with the strains 52,53,54 and WT in 5ml DSM
inoculated Germination Knock outs (B46 and B47) in 4ml LB

25.07.2016

(DS,KO,NW)

Heat shock

Spore staining

Heat shock with the stains B46 spores, B46 vegetative cells and WT spores in 1ml LB for 45 minutes at 99°, plated and put into 3ml LB to grow over night
Inoculated WT in 4ml LB Medium A
Spores were stained with Malachit greeen

26.07.2016

(DS)
The growth of the samples with heat shock only showed growth with the WT168
Inoculated the strains cotZ, cotB, cgeA, cotG in 2,5ml LB
Heat shock with the strains WT vegetative cells and WT spores (100ul in 1ml) LB for 45 minutes at 99°, plated the vegetative cells
Purification of the spores, centrifuges 10,000 × g for 10 min at 4◦C and washed with bi-distilled water 2x (the vegetative cells all died in the heat shock so we have to separate them from the spores) and put in 1ml LB

dilution row:
Prepare 3 tubes for each dilution. The first contains 900µl of DSM, the second and third 990µl each.
Add 100µl of cells (=10-1) and plate 100µl (=10-2)
Add 10µl from the 10-1 dilution to the second tube (=10-3) and plate 100µl (10-4)
Add 10µl from the 10-3 dilution to the third tube (=10-5) and plate 100 µl (10-6)
Incubate plate at 37°C and check for colonies regularly

27.07.2016

(DS)

Competent b. subtilis (WT)

plated the knock outs cotZ,cotB, cotG und cgeA
inoculated 200ul WT168 in 9ml Medium A to create competent B.subtilis cells
the samples were put in the incubator to grow. Measured the OD every 20 minutes and wait till they are past their exponential growth. That is the point t0. Now the samples stayed in the incubator for another 90 minutes. 0,45ml of pre-heated medium B were put in a tube and 0,05ml of the samples were added to it. Afterwards the samples were put in the incubator again for 90 minutes to acheive their competence.

A Glycerol stock of 168WT was made.

28.07.2016

(DS;YS)

Spore counting improved counting chaimber

inoculated the knock out strains cotZ, cotB,cotG, cgeA, B52-B56 in 3ml LB
Spores were counted with the improved Neubauer countingchamber
1. they were diluted 1 to 100 in LB (10ul sample and 990ul LB)
2. spores were counted in the counting pattern
B52: 548.556.800 spores/ml
B53: 486.400.000 spores/ml
B54: 409.600.000 spores/ml
WT168: 460.800.000 spores/ml

29.07.2016

Aliqouts

3. samples were diluted to 200.000.000 spores/ml with DSM so that there are 100 million spores per sample
B52: 4ml sample + 6,9ml DSM (548.556.800 spores /200.000.000 = 2,742 ; 2,742 x 4 = 10,9 ; 10,9 - 4 = 6,9)
B53: 4ml sample + 5,7ml DSM
B54: 4ml sample + 4,2ml DSM
WT168: 4ml sample + 5,2ml DSM

4. aliquots were made (500ul in each tube)
B52: 18 x 500ul
B53: 18 x 500ul
B54: 16 x 500ul
WT168: 17 x 500ul

Glycerol stocks were made from cotZ cotB cgeA
plated B52-56 and B49,B44,B46,B47 and cotG
inoculated B52-56, WT168 and B46 and B47 + CotZ, CotB, CotG and cgeA (from BGSC)

30.07.2016

FACS of heat shock cellls

(DS;YS;KO)
B46 and B47 were plated
B52-56,B46+47, WT168, CotZ, CotG, CotB, CgeA were incubated in the plate reader and the growth curves were measured over 6 hours

Wt168 and B46 spores and vegetative cells(400ul) were washed with PBS 1x and sonified for 15 minutes at 100%, washed with ddH2O and put in 2ml LB
Wt168 and B46 were heat shocked for 50 minutes at 99°C with 700rpm, centrifuge and put in 2ml LB
Samples were prepared for FACS (washing with PBS 1x and put in 1,1ml PBS)

1.08.2016

(DS;YS;NV)

test transformation (did not work)

test transformation was performed with the competent b. subtilis cells made on wedneyday
WT168 who has no antibiotic resistance was transformed with a backbone with chloramphenicol resistance and the vector pBS1c and plated in a selective agar with chloramphenicol
To transform from competent glycerol stocks, spin the tube at about 1600rpm for 20min, remove the supernatant (glycerol), and add 100µL of pre-warmed medium B
Mix the cells thoroughly
Add 0.6µg of DNA to the competent cells
Incubate for 30min at 37ºC with shaking
Plate 100µL of transformed cells onto selective agar

WT168 and B46 were inoculated in 5ml LB

2.08.2016

(DS,YS)

Competent WT b. subtilis

Antibiotic plates for test transformation

–> the test transformation: there is a clear growth to see on the plate, but it was a wrong concentration of Cm (too low, that is probably why the WT grew so extreme)

2 samples of WT168 were diluted to an OD of 0,1 and put in 9ml Medium A to grow
Wait till they are past their exponential growth. That is you point t0. Now you let the samples stay in the incubator for another 90 minutes. You put 0,45ml of pre-heated medium B in eppis and add 0,05ml of the samples to it –> put in the incubator again for 90 minutes. Afterwards you should have very competent cells

glycerol stocks of them:
spin down the fresh competent cells to obtain a pellet
Remove all supernatant
Re-suspend cells in 500µL 60% glycerol
Freeze tubes at -80ºC
competent b. subtilis cells: 24 samples

prepare plates for transformation: test for cm-resistance: 7ul + 1ml LB on the normal LB plates ( 11 plates )
To transform from competent glycerol stocks, spin the tube at about 1600rpm for 20min, remove the supernatant (glycerol), and add 100µL of pre-warmed medium B
Mix the cells thoroughly
Add the amount of DNA to the competent cells
Incubate for 30min at 37ºC with shaking
Plate 100µL of transformed cells onto selective agar (Cm)
samples:
1. WT
2. WT + 1ug DNA
3. Wt + 0,5ug DNA
4. Wt + 0,1ug DNA
5. Wt + 10ng DNA
6. WT + 1ng DNA
7. C.c. + 1ug DNA
8. C.c. + 0,5ug DNA
9. C.c. + 0,1ug DNA
10. C.c. + 10ng DNA
11. C.c. + 1ng DNA

B44, B49 (Julia), cotZ, cotB, cotG, cgeA (BGSC) were inoculated in 3ml LB for glycerol stocks
Glycerol stocks of cotZ and cotB were made. B44, B49, cotG and cgeA were plated.

3.08.2016

(DS)
–> test transformation did not work, there was not growth at all, probably because we did not give it enough time to recombinate
WT168 was inoculated in 3ml LB to grow for another test transformation
B44, B49, cotG and cgeA were inoculated in 4ml LB for glycerol stocks

4.08.2016

Glycerol stocks (CotT and cgeA (from Julia)

glycerol stocks of them: spin down the fresh competent cells to obtain a pellet (3minutes 16000rpm)
Remove all supernatant
Re-suspend cells in 500µL 60% glycerol
Freeze tubes at -80ºC
competent b. subtilis cells: 58 samples

WT168 war inoculated in 5ml LB

Competent B. subtilis (WT)

(DS;YS)
CotZ, cgeA (from Julia) and cotZ from BGSC were made in glycerol stock

1 samples of WT168 was diluted to an OD of 0,1 and put in 9ml Medium A to grow
Wait till it's past their exponential growth. That is you point t0. Now you let the samples stay in the incubator for another 90 minutes. Afterwards you put 0,45ml of pre-heated medium B in tube and add 0,05ml of the samples to it –> put in the incubator again for 90 minutes. You should have very competent cells now (highly motile)

5.08.2016

(DS;YS)

transformation empty back bone in WT

centrifuge the glycerol stock samples (40 x competent cells ) for 20 minutes at 14000rpm, remove the glycerol and add 100ul of Medium B
16 samples:
(DNA concentration I: 0,9 ng/ml II: 0,5ng/ml)
(Time : 1 hours, 2 hours and 5 hours ; Tempretature : 30 and 37 Degrees)
And plated on Cm plates to see if the backbone did insert

Spore purificatino (with Lysozyme )

(DS/KO)spore purification
Aliquoted spores of WT168 and B54 were taken and centrifuged for 2 mins at 13,000 x g at 25°C. The pellets were resuspended in 500ul 1xPBS, respectively. 100ul of lysozyme (15mg/ml) were added and the samples were incubated at RT for 1h. Samples were washed in 500ul 1M NaCl, 1M KCl and ddH2O, each twice. The samples were centrifuged at 13,000 xg for 1,5mins and resuspended in 1xPBS and incubated at 65°C for 1h. Samples were used for lysis (see group 2).

Growth curves in plate reader

W168, cotZ,cotB, cotG, cgeA, B44, B49, B46 and B47 were inoculated in 3ml Lb and B52-56 in 3ml Lb+cm for growth curves for 6 hours.

6.08.2016

Results from the transformation:
–> the transformation worked but due to a high amount of DNA there was growth everywhere. The controls also worked. The WT168 did not grow and the B53 did grow
Competent B.s. + 0,5ug DNA
B53 growth WT no growth

B52, B53, B55 and B56 did not grow.
Prepared 250ml (10 Plates) of Starch Agar according to Starch Agar Protocoll
W168, cotZ,cotB, cotG, cgeA, B44, B49, B46 and B47 were inoculated in 3ml Lb and B52-56 in 3ml Lb+cm for growth curves.

7.08.2016

starch plates for screening of the transformation

Plated 5 pbs1c single colonies on starch plate.
Prepared Lysozyme solution (30mg in 2ml ultra pure H2O) for Lysozyme purification. B46 and Wt168 Spores as well as their vegative cells were treated with Lysozyme solution (66µL solution in 400µL liquid culture) and incubated for one hour at 22°C. The cultures were centrifuged for 2 minutes at 13.000xg and 25°C. The pellet was resuspended in 500µL PBS. This was done twice. Afterwards the cultures were washed four times at 11700xg and after the last cycle the pellet was resuspended in 1mL PBS and send to FACS.

400µL of B46 and Wt168 spores and B46 and Wt168 vegetative cells were heat shocked for 50 minutes at 100°C. The cultures were centrifuged for 2 minutes at 11.700xg and 25°C. The pellets were resuspended in 1ml PBS and send to FACS.

WT168, cotZ,cotB, cotG, cgeA, B44, B49, B46 and B47 were inoculated in 3ml Lb and B52-56 in 3ml Lb for growth curves.
B46 and WT168 were inoculated in 6ml LB for sporulation.

8.08.2016

Growth curves

WT168, cotZ,cotB, cotG, cgeA, B44, B49, B46 and B47 were diluted to an OD of 0,17 and 200µL of each were filled into a ELISA plate with solid plastic lid. Growth curves were mesured.

aAmylase test was performed with 100µL Lugols'iodit per colony. Everything turned a dark shade of purple. (Note: Next time less Lugol's iodite and diluted and make a control).

Diluted overnight cultures of B46 and Wt168 to OD 0,1 and incubated them till OD 0,8. Centrifuge the cells at 13.000 rpm for 1 minute. Wash the pellet with bidestilled water. Resuspend the pellet in DSM (2,5ml for B46 and 4,5ml for Wt168) and let them grow over night.

Plated pbs1c single colonies on starch plate along with Wt168 colonies.

9.08.2016

aAmylase test

Did aAmylase test with pbs1c and Wt168 colonies (circa 100ul , but 2 to 1 diluted so the colour is not that saturated. The Lugol's iodite coloured the plate around the pbs1c a dark shade of purple and the colouring around the Wt168 was lighter than with pbs1c. This might mean that the transformation was succesful. Wt is capable of degrading starch, which showes itself in the lighter coloring. pbs1c is no longer capable of degrading starch and therefore showes normal colouring.
Let plate with pbs1c an Wt168 incubate further for increased starch degradation.

spore counting

Spores were counted and diluted in PBS to 200million spores per ml:
WT168: 512.000.000 and diluted in 6,24 ml PBS
B46: 588.800.000 and diluted in 3,888 ml PBS
–> made 10x B46 and 20x WT aliquots
Spore purification with lysozymes was induced (2samples from WT and B46): 1 to 6 dilution (83,3ml lysozyme and 416,66 ml samples) and in a heat block at RT for 1hour
the samples were washed with PBS for 6x and then diluted in 1ml PBS to freeze

WT, B53, B54 were inoculated in 3ml LB

10.08.2016

test with Lugol's iodine

pbs1c single colonies + WT colonies put on starch plate
add Lugol’s iodine on the colonies to see if the transformation worked
→ WT is able to reduce the starch, so there is no staining/colouring around the colonies (functionable α-Amylase)
→ the construct is not able to reduce the starch anymore so there is staining around the colonies
→ proves that the backbone got inserted in the right locus (amyE)

analysed growth curves

The growth curves were analysed:
CotG CotZ WT168

doubling time varies around:25 - 65 minutes

11.08.2016

B52-56, WT168 , B46&B47 and cgeA,cotB,cotG and cotZ were inoculated in 3ml

12.08.2016

Growth curves

B52-56, WT168 , B46&B47 and cgeA,cotB,cotG and cotZ were diluted to an OD from 0,1-0,2 and measured in the cariostar for 7hours

Sporulation was induced with the strains: Wt168, B53 and B54
–> diluted to an OD of 0,1 in 7ml LB, after 12 1/2 hours they were in their exponential growth, wash them with PBS and dulite dem in 3ml DSM

13.08.2016

Minimal medium

Made 80ml of the minimal medium for 8 plates (4 with theronine and 4 without theronine) according to protocol Culture medium

Aliquots

induced spores were harvest and treated with lysozyme (diluted 6 to 1) and incubated for an hour at room temperature, afterwards they were washed 6x with PBS and a heat shock for 1h at 65°was induced
spores were counted, diluted in PBS and aliquots with 100Million spores were made
Aliquots:
WT: 5x 500ul with 100.000.000 spores
B45: 7x 500ul with 100.000.000 spores
B53: 5x 500ul with 100.000.000 spores

14.08.2016

inoculated all the strains to make another growth curve tomorrow

15.08.2016

Growth curves

Growth curves were measured for 7 hours

sporulation was induced with the strains WT, B54 and B53

16.08.2016

new LB plates for GFP displaying strains

harvested the spores –> the samples were contaminated with small particles
B52-B56 were plated on LB plates

17.08.2016

Sporulation was induced with the stains B53, B54 and WT
cotZ, cotB, cogG and cgeA were plated and inoculated in 2ml LB

Competent b.s. cells from cotB, cotG , cotZ and cgeA

Competent cells were made from WT, cgeA, cotB, cotG and cotZ
–> inoculate the strains in LB so they have an OD600 from 0,1-0,2 and let them grow for around 4hours (till they leave the exponential growth and then an additional 90minutes
After that you add 0.05mL of the culture into 0.45mL of pre-warmed Medium B in an Eppendorf tube and incubate it for another 90 minutes at 37°C
–> you should have highly competent cells (highly motile), to make glycerol stocks, centrifuge them for 3 minutes at 13.000prm and remove supernatant
–> you have to freeze them in 60% glycerol
CotZ: 15x
CotB: 15x
CotB: 15x
CgeA: 15x
WT: 6x
Plated cotZ , cotB, cotG and cgeA

18.08.2016

Sporulation did not work –> no spores. Maybe the concentration of DSM was too high and they didn't get a chance to sporulate
Purified aliquots from WT, B53 and B46
–> (diluted 1 to 6 (1,2ml PBS and 0,2ml Lysozyme) in lysozyme and incubated them at room temperature for an hour, wash 6x with PBS)–> still chunks in there maybe from the lysozyme
WT, B53,B54 and B46 were inoculated in 3ml LB

19.08.2016

WT, B53,B54 and B46 were inoculated to grow until their exponential state (OD600 = 0,8) and then put in DSM medium to sporulate
B53 and WT168 were purified with lysozyme + washed with KCL (2x), NACL (2x) and ddh2o (2x)

20.08.2016

Test transformation (thrC)

Plates with spec antibiotic were made (30ul diluted in 1,2ml LB and distributed on 3 plates)
test transformation was performed with 50ng and 1ng DNA enmpy backbone (with spec resistance ) and competent WT168
–> 2 Glycerol stocks were zenfrifuged for 20 minutes at 13rpm to remove supernatant and add 100ul of pre-warmed Medium B
–> add DNA and incubate it at 37°C for 30 minutes /1hour + plate it on the selection medium

WT168, B53,B54 and B46 were inoculated in 3ml LB

21.08.2016

test trafo (neu)

the test transformation did not work, the antibiotic concentration was probably not right since the WT control did grow and the transformed strain did not
–> concentration of spec was too low ( standard for e.coli) we need to double it
–> try it again today and check the antibiotic dilution (this time 50ul on 400ul on each plate)
trafo:
competent b. subtilis centrifuged for 20 minutes and remove all the supernatant and add 100ul of pre-warmed medium B
add the DNA (1ng and 50ng ) and incubate it for an hour
–> plate it in selective agar

sporulation

Sporulation was induced with the strains WT,B53, B54 and B46 at 8pm

FACS

FACS with the purified spores from 19.08.2016 + 18.08.2016 and none treatet spores

22.08.2016

The induced spores were diluted with 1,2ml 1xPBS and 200ul Lysozyme to incubate for 1 hour at RT
–> afterwards they were washed in PBS one and the out in the freezer

test transformation

–> trip from Julia : spec 100ul (200ug/ml) on the plates
three competent cells were centrifuged at 13000prps for 20 minutes and the put in 100ul pre-warmed Medium B
DNA was added : 100ng DNA and in the other one 10ng
samples were incubated at 37°C for one hour and then plated in the selective agar

23.08.2016

Results from the trafo :
the plates were too highly concentrated but there was still a single colony growing on each of the plate (not the control)

24.08.2016

10 Starch plates were made according to protocol (note: add 1,5% agar) Culture medium

Transformation with the first constructs

first constructs are ready:
pBS1C pIG16_045 cgeA in cgeA k.o.
pBS1C pIG16_063 cgeA in cgeA k.o.
pBS1C pIG16_064 cotG in cotG k.o.
pBS1C pIG16_062 aGFPnano in cgeA k.o.
pBS1C pIG16_101 GST –> tomorrow

Glycerol stocks: 3x cgeA, 1x cotG and 1x WT –> centrifuge for 20 minutes at 13.000 rpm, remove supernatant and add 200 ml of pre-heated Medium B + mix thoroughly
DNA was added: 200ng DNA to each sample (not the WT168 since it is the negative control )

pBS1C pIG16_045 = 70ng/ul –> took 2,85ul for 200ng DNA
pBS1C pIG16_063 = 27ng/ul –> took 7,4ul for 200ng DNA
pBS1C pIG16_064 = 35ng/ul –> took 5,71ul for 200ng DNA
pBS1C pIG16_062 = 17ng/ul –> took 11,76ul for 200ng DNA

the samples were incubated for 1 hour and then plated on selective plates to grow (Chloramphenicol plates 1 to 5000 dilution)

25.08.2016

There is growth on each one of the plates (even the control) but it looks like e.coli (under the microscope you can see it is b. subtilis). Maybe the cm plates were too old. New plates were made and the colonies from the transformation were added to see if they grow

pIG16_045 pIG16_062 pIG16_063 pIG16_064 control

note : you have to autoclave the glycerol before you use it !!!

inoculated cgeA, cotB, cotG, cotZ and WT, B54 and B43 to grow in 2ml LB

26.08.2016

competent B. subtilis cells

The strains cgeA, cotB, cotG, cotZ and WT were grown over night and diluted to an OD of 0,1 to grow in Medium A (2ml) till they leave their exponential growth phase. After that there are incubated for another 90 minutes
Transfer 0.05mL of the samples into 0.45mL of pre-warmed Medium B in a tube and let them grow for another 90 minutes. Each tube resembles one transformation

WT: 16x
cgeA: 14x
cotB: 14x
cotG: 14x
cotZ: 14x

The samples were centrifuged and 500ul 10%Glycerol was added to freeze them in -80°C

transformation

(9am 27.8) with the constructs: pBS1C pIG16_044 cgaA 675ng DNA
pBS1C pIG16_045 cgeA 650ng DNA
pBS1C pIG16_062 cgeA 575ng DNA
pBS1C pIG16_063 cgeA 700ng DNA
pBS1C pIG16_064 cotG 625ng DNA
pBS1C pIG16_077 cotZ 600ng DNA
+WT168 as a negative control

the glycerol samples were centrifuged for 20minutes at 15000 rpm, the pallets were put in 100ul pre-warmed medium B and the DNA was added
samples were put in the shaker to grow for one hour and then were plated in selective agar (LB+Cm (1 to 5000)

The strains B53,B54 and WT were diluted to an OD of 0,1 and then grown till they reach their exponential phase (around an OD from 0,8)
The samples were washed with PBS once and then put in DSM for 24 hours to sporulate

27.08.2016

transformation

the trafo from this morning did not work (the Cml was probably too long on the plates without beeing spread –> So the concentration is wrong and the normal WT (control) can growth
another trafo with the stains:
on cm plates (7ul per 200ml per plate)
pBS1C pIG16_077 cotZ 775ng DNA
pBS1C pIG16_081 cotZ 920ng DNA
pBS1C pIG16_082 cotZ 850ng DNA
pBS1C pIG16_100 cgeA 852ng DNA
pBS1C pIG16_101 CGEa 875ng DNA

on spec plates (50ul per 200ml per plate)
pBS4S pIG16_104 cotZ ng DNA
pBS4S pIG16_105 cotZ ng DNA
pBS4S pIG16_117 cgeA ng DNA

Spores
the spores from yesterday were harvested, centrifuged and put in 4ml PBS(1x)
for the counting the spores were diluted 1 to 100
strains: WT168, B53 and B54
Spores per ml:
WT : 512.000.000 (Dilutes in 6,24ml PSB –>200.000.000 spores per ml)
B53: 435.200.000 (Dilutes in 4,70ml PSB –>200.000.000 spores per ml)
B54: 537.600.000 (Dilutes in 6,75 PSB –>200.000.000 spores per ml)

Aliquots:
WT: 21x 500ul with 100.000.000 spores
B53: 17x 500ul with 100.000.000 spores
B54: 20x 500ul with 100.000.000 spores

28.08.2016

Colony PCR

samples :
1. first colony picked from strain pIG16_062
2. second colony picked from strain pIG16_062
3. third colony picked from strain pIG16_02
4. WT (that grew on the cm plates )
5. WT (new one )
6. only primers
7. plasmid

colony PRCR with the strain iPG16_062
I) preparation of the colony + controls
1. pick a colony
2. dilute in 10ul of Tris-HCL
3. 5min. on ice
4. 1min. microwave (at full power, gloves (!) + open the samples)
5. 30 seconds on ice
6. repeat step 3 and 4 twice (so that the samples were 3 times in the microwave in the end)
7. 5min on ice

For PCR each sample gets distributed into 3 samples: the 1st one has the primers 61 + 62
the 2nd one has the primers 63 + 64
the 3rd one has the primers 57 + 58

For one sample for PCR you use:
2×0,5MM 12,5ul (Aliquots)
Primer 1 1,25ul
Primer 2 1,25ul
ddh2o 9ul
colony 1ul

the 21 samples were put in the PCR cycler with a program + afterwards transfer the samples (diluted with 2 1/2 ul 10x orange G loading dye) to the gel for 40 minutes

Control plates + antibiotics

cgeA and cotZ competent b.s. cells were zentrifuged and put in 100ul LB to grow
preparetion plates (2x):

sample	antibiotic	concentration
1.	LB	-
2.	LB + Cm	5ul with 400ul LB
3.	LB + Erm	2,5ul (10mg/ml) with 400ul LB
4.	LB + Spec	(50ul with 400ul LB)

each plate was inoculated with cgeA and cotZ and let to grow over night

WT from Julia + WT from glycerol stock were inoculated in 7ml LB to grow over night

29.08.2016

the inoculated WT (from Julia Barter (munich) and the glycerol stock were diluted in medium B to an OD600 of around 0.1 and let grown till they leave their exponential growth curve (around 4hours)and then an additional 90 minutes
afterwards the 0,05ml of the sample is diluted in 0,45ml of pre-warmed medium B and incubated for 90 minutes again at 37°C

to check the resistance of the WT and check the concentration of antibiotics we use different amount of antibiotics
(each antibiotic is diluted in 400ul LB)

Sample	Antibiotic	concentration
1.	Cml	3ul
2.	Cml	5ul
3.	Cml	7ul
4.	Erm	2,5ul
5.	Spec	25ul

this experiment was done with WT from Julia, Wt from the glycerol stock and a positive control which is CML resistance(B52)

30.08.2016

cotZ,cgeA, cotG were inoculated in 3ml LB to grow over night

Trafo

strains	Gene	Locus	Antibiotic
077	cotZ	pBS1C	7ul Cml
0811	cotZ	pBS1C	7ul Cml
082	cotZ	pBS1C	7ul Cml
100	cgeA	pBS1C	7ul Cml
045	cgeA	pBS1C	7ul Cml
062	cgeA	pBS1C	7ul Cml
063	cgeA	pBS1C	7ul Cml
064	cotG	pBS1C	7ul Cml
108	cotG	pBS4S	50ul Spec
123	mcherry in WT168	pBS4S	50ul Spec
104	cotZ	pBS4s	50ul Spec
105	cotZ	pBS4s	50ul Spec
117	cotA	pBS4s	50ul Spec
101	cgeA	pBS1C	7ul Cml

With the controls: WT on cml, cotG,cotZ,cgeA on Cml and cotG on Spec

31.08.2016

made Chloramphenicol (200ul in 1L LB) and Spectinomycin (1ml in 1L LB) plates
Spec: 40x
Cml: 40x
cgeA, cotG and cotZ cells were made competent and frozen in 10% glycerol (-80°C)
cgeA : 20x
cotG: 20x
cotZ: 18x

Transformation

strains	Gene	Locus	Antibiotic	DNA (ng/ul)	taken DNA (ng)
077	cotZ	pBS1C	Cml	51	600
081	cotZ	pBS1C	Cml	31	465
082	cotZ	pBS1C	Cml	35	494
100	cgeA	pBS1C	Cml	23	287,5
045	cgeA	pBS1C	Cml	21	203,7
062	cgeA	pBS1C	Cml	28	343,8
063	cgeA	pBS1C	Cml	48	600
064	cotG	pBS1C	Cml	19	190
104	cotZ	pBS4s	Spec	42	600
105	cotZ	pBS4s	Spec	26	299
117	cotA	pBS4s	Spec	33	429
101	cgeA	pBS1C	Cml	19	158

With the controls: WT on cml, cotG,cotZ,cgeA on Cml and cotG on Spec

01.09.2016

Starch plates

starch plates were made and the successful transformed singel colonies were transferred to confirm that the backbone is inserted in the right locus (amyE)

the medium for the competent b.s. cells (the tryptophan) was contaminated with e.coli –> throw all the competent cells out (you could see the e.coli growing on the selective medium )

inoculated new WT, cotZ, cgeA, cotB and cotG in 3 ml LB

02.09.2016

Transformation

Konstructs: 25

name	k.o.	antibiotic	DNA
044	cgeA	Cml	600ng
078	cotZ	Cml	600ng
079	cotZ	Cml	600ng
080	cotZ	Cml	600ng
085	cotG	Cml	600ng
086	cotG	Cml	600ng
088	cotG	Cml	600ng
089	cotG	Cml	600ng
096	cotB	Cml	600ng
109	cotG	Cml	600ng
077	cotZ	Cml	600ng
081	cotZ	Cml	600ng
082	cotZ	Cml	600ng
100	cgeA	Cml	600ng
045	cgeA	Cml	600ng
062	cgeA	Cml	600ng
063	cgeA	Cml	600ng
064	cotG	Cml	600ng
108	cotG	Spec	600ng
123	mcherry(WT)	Spec	600ng
104	cotZ	Spec	600ng
105	cotZ	Spec	600ng
117	cgeA	Spec	600ng
101	cgeA	Cm	600ng
122	mcherry (WT)	Erm	???
79 + 123	cotZ	Cml + Spec	300 + 241,8 ng
104 + 45	cgeA	Cml + Spec	156,4 + 300 ng

with the controls: WT on cml, spec and erm
cgeA on cml and spec
cotG on Cml and spec
cotB on Cml and spec
cotZ on cml and spec

03.09.2016

the colonies of the constructs and controls were picked and pated on starch plates to check if the backbone was inserted correctly

(Note: the controls also grew on the plates)

Since the plates were contaminated /grew the second time we through out every medium and made it again

04.09.2016

Colony PCR (KO)
A single colony was picked from following plates and inoculated in 50ul Elution buffer, respectively:
1: dCotB (grown negative control)
2: 96(GST_HA_G4S_cotB)
3: dCotG
4: 88 (GST_HA_G4S_cotG)
5: dCgeA
6: 62 (CgeA_G4S_HA_aGFPnano)
7: dCotZ
8: 79 (aGFPnano_HA_aHelix_cotZ)
The samples were heat shocked at 99°C for 10 mins and centrifuged for 30sec. The SNs were taken for following colony PCR.
As controls plasmids, wildtype B subtilis and primers were used.

the knock outs (cotZ,cotB,cotG and cgeA) were inoculated in 3ml LB from the stocks BGSC send us

05.09.2016

competent b.s. were made according to protocol Transformation of B.subtilis

06-11.09.2016

Medium was contaminated –> everything was thrown away and made new
then had some problems with the growth of competent cells
inoculated the K.O. in 3ml LB + Erm (1 to 10.000) and WT in 3ml LB

12.09.2016

tried a new protocol from munich 2012 Competent cells + transformation munich
competent cells were made from WT, cotZ, cotB, cotG and cgeA
and a transformation :

constructs	K.O.	Antibiotic	DNA ng
089	cotG	Cml	1470
064	cotG	Cml	1260
082	cotZ	Cml	900
078	cotZ	Cml	1560
101	cgaA	Cml	990
063	cgeA	Cml	1860
104	cotZ	Spec	1260
109	cotG	Spec	870
117	cgeA	Spec	1380
122	WT	Erm	1260
123	WT	Spec	810

with the control: cgeA,cotG,cotZ on Cml and Spec, WT on Erm and Spec
–> there was no growth on the controls at first , but then you could see that all plates are contaminated with e.coli and there was a clear growth after a few hours
–> there was growth of b. subtilis on the plates with 122 and 123 (the mcherry constructs)

13.09.2016

Trafo

(with K.O.)

constructs	DNA ng	K.O.	Antibiotic
088	930	cotG	Cml
086	1230	cotG	Cml
085	1020	cotG	Cml
081	810	cotZ	Cml
079	720	cotZ	Cml
077	1140	cotZ	Cml
100	960	cgeA	Cml
062	1260	cgeA	Cml
044	1170	cgeA	Cml
045	960	cgeA	Cml
108	750	cotG	Spec
105	720	cotZ	Spec

with the controls : cotG and cot Z on Spec and Cml, cgeA on Cml and WT on Spec

14.09.2016

New minimal medium , Cml and Spec plates were made

Transformation

another transformation was performed with all constructs:

constructs	K.o.	Resistance
44	A	Cml
45	A	Cml
62	A	Cml
63	A	Cml
64	G	Cml
77	Z	Cml
78	Z	Cml
79	Z	Cml
80	Z	Cml
81	Z	Cml
82	Z	Cml
85	G	Cml
86	G	Cml
88	G	Cml
89	G	Cml
96	B	Cml
100	A	Cml
101	A	Cml
104	Z	Spec
105	Z	Spec
108	G	Spec
109	G	Spec
117	A	Spec
122	WT	Ery
123	WT	Spec
112	B	Spec

+Controls –> there was no growth

15.09-23.09.2016

- made MNGE medium + Plates for the constructs from polen (for characterazition of a biobrick)
- Transforamtion of the constructs 33,34,35

24.09.2016

Transformation with 1000ng DNA

Stain	contrains	K.O.	Selectionsmedium
126	mcherry	WT/cgeA	Spec
128	antiGFPnano	cotaB	LB (no selection marker)
129	mcherry	cgeA	MNGE
141	GST	cgeA	MNGE
131	GST	cotZ	MNGE
150	antiGFPnano	cgeA	Cml
151	antiGFPnano	cotG	Cml
159	antiGFPnano	cotZ	Cml
160	antiGFPnano	cotZ	Cml
157	GST	cotZ	Cml

+controls on Spec,Cml and MNGE

–> there was no growth on the plates, possible a too high amout of DNA (could be toxisch)

25.09.2016

Strain	K.O.	construct	Antibiotic
141	cgeA	polenconstruct	Cml
131	cgeA	polenconstruct	Cml
150	WT/cgeA	antiGFPnano	Cml
151	WT/cgeA	antiGFPnano	Cml
159	WT/cotZ	antiGFPnano	Cml
160	WT/cotZ	GST	Cml
157	WT/cotZ	antiGFPnano	Cml

–> with neg. controls
- only use 600ng of DNA

26.09.2016

–> the tansformation worked with the strains on the Cml plates –> colonies were picked and plated on new Cml plates and starch plates to confirm that the bakcbone got inserted in the right locus (AmyE)

Transformation

stains	K.O.	Antibiotic
131	cotZ	MNGE
141	cgeA	MNGE
126	cgeA/WT	Spec
142	cotZ	MNGE
034	WT	MNGE

27.09.2016

–> the stains on the starch plates did not reduce the starch, that confirms that the constructs are inserted correctly
–> picked colonies to inoculate them over night

Transformed constructs:

strains	construct	transformed in
151	antiGFPnano	in WT and cotG
157	antiFPGnano	in Wt and cotZ
160	GST	in Wt and cotZ
150	antiGFPnano	in Wt and cgeA
159	antiGFPnano	in Wt and cotZ

28.09.2016

- induced sporulation with the 10 succsessful constructs
- inoculated the knock out strains cgeA,cotB,cotG, cotZ and Wt

29.29.2016

- induced sporulation with the negative controls (A,B,G,Z and WT)
- another transformation with different strains on MNGE-medium
- harvesting the spores of our constructs:
1. 160 Wt
2. 160 cotZ
3. 151 cotG
4. 151 Wt
5. 150 Wt
6. 150 cgeA
7. 159 cotZ
8. 159 Wt
9. 157 cotZ
10. 157 Wt
the spores were purified (treated with lysozyme: 1:6 dilution and incubated for 1hour at RT and washed with 1xPBS 6 times), counted and aliquot were made with 100million spores per 500ul

30.09.2016

- Spores from the negative controls were harvested, purified, counted and aliquot were made
- strains were inoculated in LB

1.10.2016

- spore purification of all constructs and controls for the westernblot (group 3)

2.10.2016

- inoculated the 10 strains in LB+ Cml
- induced sporulation with negative controls (Wt, A,B, G, Z)

3.10.2016

- purification of the Wt,B, A, G, Z +the constructs 1&2 (for the GST essay from group 5)

4.10.2016

- spore purification of all 10 constructs

Transformation

strain	construct	K.O.	Antibiotic
156	GST	G & WT	Cml
170	antiGFPnano	G &WT	Cml
166	antiGFPnano	A &WT	Cml
155	antiGFPnano	Z	Cml
160	GST	Z	Cml
169	antiGFPnano	G & WT	Cml
167	GST	G & WT	Cml
153	GST	Z & WT	Cml
152	GST	A & WT	Cml
168	GST	A & WT	Cml

5.10.2016

Glycerol stock were made:
pIG16_175 (160 Z)
pIG16_176 (160 WT)
pIG16_177 (151 G)
pIG16_178 (151 WT)
pIG16_179 (150 A)
pIG16_180 (150 WT)
pIG16_181 (159 Z)
pIG16_182 (159 WT)
pIG16_183 (157 Z)
pIG16_184 (157 WT)

- Spore purification of all constructs and negative controls for a westernblot of group 3 (700 Milllion purified spores per sample)

–> the transformation was successful, the strains grew on the Cml-plates when inserted in the WT
–> picked colonies on starch plates to check if the backbone is inserted into the right locus

6.10.2016

- starch plates were checked –> most of the colonies didn't reduce the starch which means that the backbone was inserted correctly
- → picked colonies were inoculated :
156 WT GST
167 WT GST
168 WT GST
153 WT GST
152 WT GST
- neg. contgrols were inoculated
- all 10 constructs were inoculated

7.10.2016

- 19 samples (10 old constructs, 5 new GST constructs and the 4 negative controls)were diluted in 10ml LB to an OD from around 0,1
- when they reach their exponential growth they were put in DSM to sporulate for 24 hours
- spores were harvested, purified, counted and aliquots were made

Team:Freiburg/NotebookSpores

Our Protocols

starch plates for screening of the transformation