15/05/18

PCR

PCR	PCR Mix 1	PCR Mix 2
GXL buffer	10 uL	10 uL
dNTPs (from GXL kit	4 uL	4 uL
GXL polymerase	2uL	2uL
ICD001 (10 uM)	0.5uL	1uL
ICD007/ICD008 (10 uM)	0.5uL	1uL
EC93 genomic DNA (15 ng/uL)	0.5 uL	1 uL
ddH2O	32.5uL	29.5uL

Program	time
Start cycle (30x)
98 °C	10 s
60 °C	15 s
68 °C	2 min
close cycle
8 °C	store

Ran of analytical gel. No bands were observed.

15/05/19

PCR

Bands visible! Lanes 1 and 2 right of the ladder should have a 10506 bp band. Lanes 3 and 4 right of the ladder should have a 11414 bp band (gel description should read EC93 instead of EC90).
PCR cleanup (according to Thermo Scientific protocol), elution in 21 ul.
Concentrations according to Nanodrop: EC93 w/ 001+007: 92 ng/ul; EC93 w/ 001+008: 57 ng/ul

15/05/21

PCR

Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.

PCR	PCR Mix 1
GXL buffer	10 uL
dNTPs (from GXL kit	4 uL
GXL polymerase	2uL
ICD0014 (10 uM)	1 uL
ICD0015 (10 uM)	1uL
1:10 diluted plasmid X	1 uL
ddH2O	31 uL

Program	time
Start cycle (30x)
98 °C	10 s
60 °C	15 s
68 °C	35 s
close cycle
8 °C	store

Turned out that the template was wrong. Inoculated 3 ml LB+chloramphenicol for overnight culture of correct Curly clone (clone No. 3) for Miniprep tomorrow.

15/05/22

PCR, Gibson Assembly and Transformation

Miniprep of 1.5 ml of Curly clone No. 3 according to Thermo Scientific Protocol.
PCR with primers ICD014 and ICD015 and freshly prepped Curly plasmid to amplify the backbone for the CDI plasmid (pICD001). Reaction mix and PCR program see 15-05-21.
Analytical gel: Correct size! (3391 bp)

Proceeded with PCR clean up with subsequent Nanodrop measurement:
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008) G.A. reaction mix:

Gibson mix	volume
Insert	2.65ul (= 151ng = 0.02 pmol)
Backbone	0.54ul (= 45ng = 0.02 pmol)
G.A. master mix	10 uL
ddH2O	6.81 uL

G.A program:

temperature	time
50 °C	15 min
16 °C	store

Electroporation of Gibson assembly mix with NEB turbo
• Thaw NEB turbo cells on ice
• Dilute G.A. mix 1:3 (5ul mix + 10ul ddH2O)
• Add 2ul of dilution to 25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate

15/05/26

PCR, Gibson Assembly and Transformation

On 15-05-22 DpnI digestion has been forgotten, so the transformed cells probably carry the Curly plasmid instead of the Gibson assembly product. So we repeated the procedure including DpnI digestion.
PCR of Curly backbone: See 15-05-21
DpnI digestion program:

temperature	time
37 °C	30 min
16 °C	store

(No enzyme deactivation was carried out)
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008) G.A. reaction mix:

Gibson mix	volume
Insert	2.65ul (= 151ng = 0.02 pmol)
Backbone	0.51ul (= 45ng = 0.02 pmol)
G.A. master mix	10 uL
ddH2O	6.84 uL

G.A program:

temperature	time
50 °C	15 min
16 °C	store

Electroporation of Gibson assembly mix with NEB turbo
• Thaw NEB turbo cells on ice
• Add 2ul of Gibson assembly product to25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate (300µL of 0,4% Glucose added to the plate to reduce transcription of Lac promoter)
Gibson mix was not diluted 1:3 in contrast to first attempt on 15-05-22

15/05/27

Plasmidisolation

Colonies are grown wich hopefully carry CDI plasmid. To check this, 12 colonies have been picked and cultivated in 3mL LB broth with additional 0.4% glucose.
In the evening, 1.5ml of cell suspension were pelleted and Miniprep was carried out with the Qiagen Robot.

15/05/28

Plasmidisolation, SacI Digest

The concentration of all the 12 colonies was measured and noted as follows:

plasmid	concentration ng/uL
#1	77
#2	30
#3	90
#4	88
#5	88
#6	86
#7	97
#8	74
#9	84
#10	88
#11	62
#12	81

A mastermix for all plasmids was made:

Digest	volume
DNA template	10 uL
Cutsmart buffer	2 uL
Distilled water	7 uL
SacI	1 uL
total	20 uL

all the colonies were then incubated for 1hr at 37 °C on an thermoblock
Analytic gel ; correct size abt 6700 and 8000

Clones 1 to 11 show the expected band pattern. We send out the abt 50ul of the 1st and 3rd colony for sequence reading.
The DNA sample was send to determine the sequence and it was positive. We actually got the sequence which we expected.

15/05/31

Overnightcultures

Inoculation of • four BBa_I13522 (GFP) clones in 3ml LB+CAM overnight
• Olga's W3110 strain with integrated RFP and Kann resistance in 3ml LB+Kann
• W3110 wildtype in 3 ml LB as host for our GFP construct

15/06/1

Transformation into chem. comp. cells

Plasmid purification (four BBa_I13522 (GFP)) was realized using the "Thermo Scientific GeneJET Plasmid Miniprep Kit"
Tansformation: GFP_1 Plasmid into E.coli (strain W3110)
pICD001-1 Plasmid into E.coli (strain W3110 with integrated RFP)
pICD001-3 Plasmid into E.coli (strain W3110 with integrated RFP)
Transformation protocol: 1. Dilute 1:100 overnight culture (Max 15-05-31)
2. Grow 10 mL day culture (2.5 h) at 37 °C
3. Take 1 mL, spin down full speed (1 min, 17.000 rpm), put on ice
4. Add to pellet 100 micro liter of TSS, resuspend on ice.
5. Add 1 micro liter of Plasmid (GFP_1 ; CDI_1; CDI_3)
6. Incubate 45 Min on ice
7. Put at 42 °C for 1 min.
8. Incubate 15 min on ice
9. Add 1 mL LB Medium with 0.4 % Glucose, resuspend, incubate 2 h incubator at 37 °C, 220 rpm
10. Spin down, resuspend in 50 micro liter residual media, plate on LB-Kann, incubate at 37 °C over night (step 10 done by Max)

15/06/2

Sequencing results

Colonies on all three transformation plates!

• GFP plasmid (BBa_I13522): Colonies beyond count
• pICD001-1 Plasmid into E.coli (strain W3110 with integrated RFP): 23 colonies
• pICD001-3 Plasmid into E.coli (strain W3110 with integrated RFP): 26 colonies

Finally the sequencing of pICD001-1 and pICD001-3 is done! Correct Gibson assembly sequences were checked by sequenceing with standard primers iGEM130-fwd and iGEM160-rev and 100ng/ul pICD001 plasmids. Both plasmids are correct, so pICD001-1 will become pICD001 and pICD001-3 can be trashed.
As a result, the W3110/RFP strain transformed with pICD001-1 will become strain CDI002 (a.k.a. the killer strain). The W3110 wildtype transformed with the GFP plasmid (BBa_I13522) will become strain CDI003 (a.k.a. the Opfer strain).
Inoculation of CDI002 and CDI003 in 3 ml LB CAM +1%glucose

15/06/3

IPTG dose response

Dose-response curve of CDI002 at different induction levels --> if Cdi operon is expressed, we expect a higher metabolic burden at high induction compared to low induction. The used broth was LB with 1:1000 Chloramphenicol and 1 % glucose. The concentrations of IPTG were gained by dilution series.

15/06/5

IPTG dose response, killing assay

Repetition of Dose-response assay with higher IPTG concentrations. At first the overnight cultures of 4 strains (W3310, W3310-RFP, CDI 002, CDI 003) were diluted to an OD600 of 0,08 to a volume of 10mL LB-medium

Abnormalities/interpretation:
• Flask with CDI003 with 1,6µM IPTG fell over. So last measurement was after 190min.
• CDI003 (GFP strain) grew fast at the beginning. After 3,5h the cells clumped together and the medium cleared up.
• The influence of different IPTG concentrations on the growth rate is very low but a slight trend is noticable.

Preparing of daycultures for flow cytometry in different broths.
Broths:
LB = LB-medium with 0.8 mM IPTG
LB-CAM = LB-medium with 0.8 mM IPTG and 1:1000 Chloramphenicol

culture (broth, Time [min]	0 min	60 min	90 min
LB, W3110 RFP	0.1	0.25	0.4
LB, W3110	0.1	0.41	0.7
LB-CAM, CDI002	0.1	0.21	0.46
LB-CAM, CDI003	0.1	0.32	0.59
LB,CDI002	0.1	0.23	0.45
LB,CDI003	0.1	0.31	0.65

The different cultures were diluted to 0.4 [OD-600] and IPTG was added to a constant overall concentration.
Different cultures were mixed (2 mL/ 2mL) to a total volume of 4 mL, incubated at 37 °C, 250 rpm for 3 hours. An Aliqout of 0.5 mL was mesured every 60 Minutes.

number	mixed cultures	ratio	broth
1	CDU002+CDI003	1:10	LB
2	CDU002+CDI003	1:1	LB
3	CDU002+CDI003	10:1	LB
4	CDU002+CDI003	1:10	LB-CAM
5	CDU002+CDI003	1:1	LB-CAM
6	CDU002+CDI003	10:1	LB-CAM
7	W3110 RFP+CDI003	1:10	LB
8	W3110 RFP+CDI003	1:1	LB
9	W3110 RFP+CDI003	10:1	LB
10	Control CDI002 only		LB
11	Control CDI003 only		LB
12	Control W3110		LB
13	Control W3110 RFP		LB
14	Control CDI002 only		LB-CAM

Observations:
The victim strain CDI003 is overgrown by CDI002, but also does the W3110 RFP strain.
The victim strain CDI003 starts shrinking after reaching of an OD-600 of 1.2.
Aggregates in samples 1,7,11 and 8
Optical whitening in samples 11 and 12
FACS results (data not shown) were not as expected so we will repeat the experiment.
Experiment will be repeated with changed parameters: lower density, lower flowrate, other broth, change of intensity, change of voltage, different FACS parameters.

15/06/9

PCR, DpnI Digest, Overnightcultures

PCR of pCDI001 (full length CDI operon) with Primers iCD003 and iCD007. Result is linearised plasmid withour CdiA-CT and CdiI.
PCR progam and Mix with GXL Polymerase are listed in the protocol section.

A DnpI digest was done afterwards.
Inoculation of 10 W3110-RFP clones in 3 ml LB over night to check with FACS the next day (last FACS measurement showed three populations in mCherry histogram. We wanted to check, if this is clone-specific or the same for different clones).
Inoculation of W3110-WT, W3110-RFP, CDI002 and CDI003 in 3 ml LB and TB to compare overnight growth tomorrow.

15/06/10

PCR Purfication, Bluntend cloning

Sample Purification
The Sample DNA was purified with the purification kit of GeneJET PCR
to the 49ul volume of sample was added 49ul of binding puffer, mixed, centrifugated, 700ul wash Buffer added ,centrifugated for one minute twice to assure the removal of any residual of wash buffer. 50ul Elution Buffer was then added to the column containing the DNA sample and centrifuged. A concentration of 65,51ug/ml was measured at the nanodrop. Blunt-end cloning Blunt-end cloning of the purified PCR fragment was done as follows and at 37°C incubated for 30 min 17ul sample DNA 2ul T4 Ligase Puffer 1ul Kinase (T4-PNK) 20min inactivation of T4-PNK at 65°C Made LB plates with CAM and 0.8 mM IPTG

strain	LB media	TB media
W3110-WT	6.6	3.1
W3110-RFP	5.8	2.8
CDI002	4.1	1.9
CDI003	4.1	1.4

-> Strains grow roughly twice as dense in LB than in TB
-> Strains harboring a high copy plasmid (CDI002 and 003) grow only half to 2/3rd as dense as strains without plasmid
FACS result of yesterday's o.n. cultures: All show the high mCherry peak in histogram, not the intermediate one or the "negative" peak. The occurence of different mCherry populations seems to be cell cycle specific and not clone-specific.

15/06/11

swarming assay; Transformation

To get a better negative control than the W3110-RFP for our swarming plates and flow cytometry mesurements, we transformed blunt-end-cloned fragment (plasmid without CdiA-CT and CdiI (pICD002)) (made by Daniel 15-6-09) into W3110-RFP strain. Following Protocol was used:
Transformation of non-competent E.coli cells
To see how much Plasmid we need to get colonies on LB+CAM plates we used different amounts of Plasmid for the Transformation Protocol.

• 1. 0.1 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002)
• 2. 1 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002)
• 3. 10 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002)

15/06/12

swarming assay; Transformation

On all LB+CAM plates colonies were observed. Most colonies resulted from the third Transformation with 10 µL Plasmid. Six colonies were picked for overnight cultures to perform Miniprep the next day.
Swarming assay
A swarming assay was performed to check if growth inhibition can be proven. LB-Plates (CAM, 0.8mM IPTG) with 0,27% Agar were used to give the bacteria the possibility to swarm across the plate.
After 3 days incubation at 30°C the plates were put on a blue light screen to visualise fluorescence.

The result is that the bacteria stopped to grow a short distance before getting in contact with each other (Green: no fluorescence: "killer strain" CDI002; Green: "target strain" CDI003). This might be due to quorum sensing or nutrient depletion.

15/06/13

Plasmid isolation

Miniprep of the six pICD002 candidates according to Thermo Scientific protocol.
Undigested pICD002 candidates ran on analytical gel

15/06/16

Overnight cultures, sequencing

Overnight cultures of CDI002, CDI003, W3110, NEBTurbo with pICD001
Plate CDI002, CDI003 and CDI004 each on a third of a LB-Plate
Sent pICD002-1 and pICD002-6 for sequencing with reverse primer 160 that binds downstream of CdiI and the CdiA-CT that we want to kick out.

15/06/17

growth analysis, SDS-Page

Diluting of all overnight cultures to an OD600 of 0,05 to start a day culture OD600 with time:

strain	T 0	2 hours	5 hours
W3110	0.05	0.69	3.1
CDI002	0.05	0.10	0.20
CDI003	0.05	0.07	0.17
CDI004	0.05	0.14	0.64

After 5 hours the growth of CDI002, CDI003 and CDI004 was to weak to start competition assay so no co-culture assay was started. To find out why day cultures did not grow properly we checked the overnight cultures under the microscope with no result.
Day cultures are diluted 1:100 and plated on LB-Agar plates to have single colonies on the plates.
SDS-PAGE was started to see if CdiA, CdiB and CdiI are expressed. All steps are done with CDI002 and W3110 as control. Sepperating gel:

Chemicals	volume	volume
Acrylamide percentage	6	15
water	5.2 mL	2.2 mL
Acrylamide_Bis-acrylamide (30%;0.8%)	2 mL	5 mL
1.5M Tris (pH=8.8)	2.6 mL	2.6 mL
10% SDS	0.1 mL	0.1 mL
10% ammoniumpersulfat	0.1 mL	0.1 mL
TEMED	0.01 mL	0.01 mL

Stacking gel:

Chemicals	volume
water	2.975 mL
0.5 M Tris-HCl, pH6.8	1.25 mL
10% SDS	0.05 mL
Acrylamide/Bis-acrylamide (30%; 0.8%)	0.67 mL
10% Ammoniumpersulfat	0.05 mL
TEMED	0.005 mL

Neither CdiA nor CdiI nor CdiB could be observed on the SDS page (data not shown).
1mL of cells are centrifuged and supernatant is discarded.
Cells were prepared in different ways:

• 1. Pellet is resuspended in 50µL of sample buffer.
  Samples are mixed by pipetting up and down for 2 minutes to break CDI A if present on the surface of the cell, suspension is centrifugated and supernatant is used for gel
• 2. Cells are used whole.
  65µL of sample buffer is used to resuspend the pellet, cells are incubated for 10 minutes at 96°C, centrifugation for 3 minutes at max speed.
• 3. Cells 50µL of lysis buffer is used to resuspend the pellet, 2µL of protease inhibitor is added, incubation at room temperature for 10 minutes to lyse the cells, centrifugation for 10 minutes at max speed and 4°C
  • a. Supernatant is used with 16µL SDS-buffer
  • b. Pellet is used with 65µL sample buffer

We found out that the W3110-RFP strain that we were supplied with still harbors a plasmid that contains GFP and kan-resistance! We have to make strain CDI002 lose that plasmid before we do any other assay: Plate 6 clones on fresh CAM-plate every day and transfer that culture onto kann-plate as negative control. Only if there is no more growth on the kan-plate we can continue! All FACS data acquired so far is useless, because with CDI002 being positive for GFP and mCherry we cannot make a proper analysis.
Sequencing results of pICD002-1 and pICD002-6: Nice sequence up to the blunt-end cloning site! Then multiple sequences seem to overlap. Cloning needs to be repeated!

15/06/18

growth analysis, SDS-Page

Repeated the PCR for pICD001 backbone without CdiA-CT and CdiI (see 15-06-09) and ran analytical gel:

DpnI digest of the PCR mix to get rid of methylated pICD001 template DNA
(This time DpnI was heat inactivated, just to be sure that it does not interfere with the following steps)
PCR cleanup using Thermo Scientific Cleanup kit. We eluted in 30 ul pre-warmed elution buffer (recommended for fragments >10 kb).
Nanodrop: 99ng/ul DNA

15/06/19

blunt end cloning; ligation

5'-phosphorylation of PCR fragment with polynucleotide kinase (for subsequent blunt-end cloning). PNK mix:

Chemicals	volume
DNA template	17 uL (0.184 pmol)
T4 DNA ligase buffer	2 uL
PNK	1 uL

Program:

temperature	time
37 °C	30 min
65 °C	20 min

Use 0.02 pmol DNA for ligation --> 2.56 ul of PNK mix Ligation mix:

chemicals	volume
ddH20	14.44 uL
PNK mix	2.56 uL
T4 DNA ligase buffer	2 uL
T4 DNA ligase	1 uL

Program:

temperature	time
RT	2 h
65 °C	10 min

15/06/24

plasmidisolation; Digest

Miniprep of the six pICD002 candidates according to Thermo Scientific protocol. Elution in 30µL elution buffer
We wanted to check if all pICD002 candidates got the right plasmid formation. So we did an Restriction assay with the six pICD002 candidates:

Restriction assay	pICD002_1	pICD002_2	pICD002_3	pICD002_4	pICD002_5	pICD002_6
DNA	1.35 mL	1.63 mL	3.33 mL	2.77 mL	2.13 mL	1.36 mL
NEB: cutsmart buffer	2 mL	2 mL	2 mL	2 mL	2 mL	2 mL
BSTZ17I (restriction enzym)	0.5 mL	0.5 mL	0.5 mL	0.5 mL	0.5 mL	0.5 mL
EcoRI (restriction enzym)	0.5 mL	0.5 mL	0.5 mL	0.5 mL	0.5 mL	0.5 mL
water	15.65 mL	15.37 mL	13.67 mL	14.23 mL	14.87 mL	15.64 mL

Candidates 1 and 6 show the expected band pattern (although we forgot to load a marker). Those DNA samples were send to determine the sequence using following mix:
Volume: 15 µL
100 ng DNA/µL
2 µL pICD002_X (1 and 6)
2µL Primer
11 µL Water

15/06/25

growth experiment

Due to our results this far we decided to go mesure a new growth curve (OD600 ) with CDI001 (W3110 + pCDI001) and W3110: used browth: LB + 0.2 % Glucose and Chloramphenicol for CDI001 to hold the selection pressure

time/strain	W3110	CDI001
0 min	0.1	0.1
40 min	0.2	0.12
90 min	0.71	0.18
120 min	1.13	0.24
180 min	1.0	0.43
230 min	1.1	0.72
270 min	1.0	1.0
320 min	1.2	0.6
360 min	1.2	0.43
420 min	1.2	0.43
480 min	1.3	0.44

After 120 minutes we add IPTG to a total concentration of 0.5 mM (flask with W3110)
After 270 minutes we add IPTG to a total concentration of 0.5 mM (flask with CDI001)
After an OD-600 of 1.0 was observed we aliqout the equivalent of 1 mL of cells at OD600 = 1.0 in a 1.5 mL microfuge tube (t=0)
Next step was induction with IPTG (0.5 mM) and continued shaking at 250 rpm for serveral hours
Aliquots were extracted every hour.
SDS Gel data here not shown.Growth of strains harboring plasmids is still very poor. We came to the conclusion that maybe not the plasmids (and the metabolic burden that is connected with them) are the problem, but the medium. Maybe the wrong antibiotic was added to LB. --> Prepared fresh medium for growth curves next week.

15/06/29

low copy plasmids

Sequencing result of blunt-end ligation: pICD002-6 has 3 deletions that destroy the stop codon that was supposed to be introduced, so this cannot be used. PICD002-1 also has a two-base pair deletion at the site of ligation but that does not destroy the stop codon, nor does it destroy the SpeI site of the iGEM suffix sequence. Not an ideal result but something that is good to work with! --> pICD002-1 becomes pICD002.
To reduce metabolic burden for the cells, we plan to switch to low copy plasmids, namely pSB3C5 and pSB4C5. They were both available in the Spring 2015 distribution with the same insert (BBa_J04450, a lac-inducible RFP gene). --> both were transformed with DH5alpha (see: Transformation into chemocompetent cells).

15/06/29

plans

Planned growth curve experiment was aborted due to growth problems of cultures with plasmid.

15/07/01

platereader

To check if the growth problems are depending on the antibiotic concentration or if the growth is different if the bacteria are taken from plate or from an overnight culture a growth curve was performed in a 24 well plate. 1,5mL of Medium (either with or without chloramphenicole (see table)) is inocculatet with a colonie from plate or with 10µL overnight culture (OD around 7,00)
The results are shown in the following growthcurve:

15/07/02

sequencing

Sequencing results: PSB3C5-1 and -2: Both correct! --> pSB3C5-1 will be used from now on
PSB4C5-1: Forward primer sequencing failed, reverse seqencing looks correct except for point mutation in lac promoter BBa_R0010€€
PSB4C5-2: All correct except for same point mutation as in clone 1: A to G in position 117 of BBa_R0010. Since both clones have the mutation we assume that it was already in the registry plate and will use pSB4C5-2 for further experiments.

15/07/03

clean streak

Clean streak of pSB3C5 and pSB4C5:
Under the magnifier we saw colonies that were half red and half white. Due to lacI on a bacterial artificial chromosome all colonies were expected to be white.

15/07/06

cloning

We want to improve our pICD001.To accomplish this goal we decided to put 6 nucleotides between the ribosome binding site and the start of the CDI_A. For the PCR conditions and DpnI digest see protocols.
PCR product purification was accomplished with QiAquickPCR Purification Kit (50)
To create a new plasmid we did a Gibson assembly with the linear PCR product:
used DNA 100 ng --> 3 µL
add 10 µL of Gibson Assembly Mastermix (2x)
add dd Water to a total volume of 20 µL
incubate 37 °C for 20 min
The product was transformed into electrocompetent cells (see protocols).

15/07/07

growth problems, cloning and platereader

Transformation was succesful. To check if the additional 6 bp were added between promoter and ribosome binding site, pICD007 was prepped with Thermo Scientific miniprep kit and eluted in 30µL.
One more idea concerning our growth problems is that the chloramphenicole molecules are degraded during the first hours of an overnight culture and the plasmids are lost at the end of the overnight culture. So finally there could be a lot of living cells but only a few carry the plasmid. If this culture is used to inocculate the day culture with new chloramphenicole most of the cells die immediately.
To check this theory, we started a new platereader experiment using different concentrations of chloramphenicole. The next day we plan to plate these overnight cultures each to LB and to LB with chloramphenicole to check if the cells are dead after overnight or if they just lost the plasmid
We want to change our CDI operon from a high copy plasmid into a mid and low copy plasmid (5 and 15 copys per cell) therefore we used PCR to amplify the CDI operon, the plasmid backbone from pSB3C5 and pSB4C5 (5 and 15 copys per cell) with 3850 basepairs and 4300 basepairs. Also we amplified lacI with 1500 basepairs. In another experiment we wanted to create an polycistronic plasmid with mCherry therefore we amplifyed most of the backbone from pSB3C5 (3800bp), pSB4C5(4300bp) and again the CDI operon without RBS.
We used the following primers and templates:

fragments	fragment 1	fragment 2	fragment 3	fragment 4	fragment 5	fragment 6	fragment 7
template (1:100)	pSB3C5-J04450	pSB4C5-J04450	pICD001	pICD001	pSB4C5-J04450	pSB4C5-J04450	pICD001
forward primer (10 uM)	ICD0034	ICD0034	ICD001	lacI, AaII rev	ICD0036	ICD0036	ICD0038
reverse primer (10 uM)	ICD0035	ICD0035	ICD008	ICD0033	ICD0037	ICD0037	ICD0039
desired fragment length	3850 bp	4300 bp	11500 bp	1500 bp	3800 bp	4300 bp	11500 bp

Protocol Standard PCR and DpnI digest.

15/07/08

gibson assembly, plating of platereader wells

The cultures of wells A2, A3, A4, D2, D3, D4 are diluted 1:100 twice. 20µL are plated each on LB and LB+CAM Agar plates
For the gibson assembly to creat our new plasmids we need to purificate our PCR products. Purification was accomplished with QiAquickPCR Purification Kit (50)
Transformation
Plating of cultures from plate reader:
The cultures of wells A2, A3, A4, D2, D3, D4 are diluted 1:100 twice (--> 1:10000).
20µL are plated each on LB and LB+CAM Agar plates. This way we are trying to find out how many cells are actually alive in our overnight culture and how many of those still have our plasmid. Only if we have roughly the same number of colonies on the LB+CAM plate as on the LB-only plate, we can be sure that all cells still harbor the plasmid.
G.A. reaction:

fragments	volume
Fragment 1	0.76 uL (=36.7 ng = 0.14 pmol)
Fragment 3	1.88 uL (=100 ng = 0.14 pmol)
Fragment 4	0.32 uL (=13.8 ng = 0.14 pmol)
G.A. master mix	10 uL
ddH2O	7.04 uL

fragments	volume
Fragment 2	0.68 uL ( 40.9 ng = 0.14 pmol)
Fragment 3	1.88 uL (=100 ng = 0.14 pmol)
Fragment 4	0.32 uL (=13.8 ng = 0.14 pmol)
G.A. master mix	10 uL
ddH2O	7.12 uL

fragments	volume
Fragment 5	0.60 uL (=36.7 ng = 0.14 pmol)
Fragment 7	2.22 uL (=100 ng = 0.14 pmol)
G.A. master mix	10 uL
ddH2O	6.84 uL

fragments	volume
Fragment 6	0.99 uL (=40.9 ng = 0.14 pmol)
Fragment 7	2.22 uL (=100 ng = 0.14 pmol)
G.A. master mix	10 uL
ddH2O	6.84 uL

used Protocol Gibson assembly.

15/07/09

results from platereader

Counting of the colonies from the overnight plate reader experiment:
Numbers are: Colonies per CAM plate / colonies per LB plate
(The CAM-plates had the SAME antibiotics concentration (34 ug/ml) ONLY the media in the plate reader experiment had varying concentrations.)
Outcome:
• CDI002 (harbors high copy plasmid, ca. 200 copies per cell) needs high CAM concentration to keep up the selection pressure. Otherwise cells lose the plasmid.
• DH5alpha with pSB3C5 (10 - 12 copies per cell) counterintuitively seems to lose the plasmid at high chloramphenicol concentrations, whereas the cells seem to keep it at low concentrations
• Same applies to DH5alpha harboring pSB4C5 (ca. 5 copies per cell). This however grows much worse.

15/07/14

SDS-Page

Preparing of SDS-Page samples for pCDI007: We wanted to see the expression of the CDI operon with the new spacer sequence between RBS and startcodon. Result of the SDS page (data not shown): no signals form any Cdi operon protein can be found. Maybe we need to change the strain or use a lowcopy plasmid in oder to decrease the metabolic burden.
Samples of the overnight culture were plated out on LB Agar and LB Agar + Chloramphenicol plates. To see if our E.coli cells lose our Plasmid. On both plates we counted equal colonies.
Test Digest of pCDI003_1, pCDI003_2, pCDI003_3, pCDI004_1, pCDI004_2, pCDI004_3, pCDI005_1, pCDI005_2, pCDI005_3, pCDI006_1, pCDI006_2,pCDI006_3.
Result of this test digest (picture not shown) all plasmids show signals in not exspected areas. To gain the new constructs we will do the Gibson assemblys again.
Gibson assembly: Protocoll 15-07-08.

15/07/15

SDS-Page

The transformants (pICD003=reaction 1, pICD004=reaction 2, pICD005=reaction 3, pICD006=reaction4) of the Gibson assembly are streaked out on Agar-Plates and from the same colonies some cells are used to inocculate overnight cultures for Miniprep.
We wanted to see if our SDS-Pages from pCDI007, pCDI002 and the wildtyp differ from each other. We obtain no difference between the aliquots of the different constructs . So far we only can see expression of the chloramphenicol resistance protein.

15/07/16

Plasmidisolation

Miniprep of pICD003, pICD004, pICD005 and pICD006 according to Thermo Scientific protocol. Elution in 30µL elution buffer
Test digest: To see with which plasmid we want to work form here on we used a test digest to get further information about our gained plasmids.
We used the restriction enzyms EcoRI and fragments with 6 kBp and 8 kBps were expected.
So far fragments were obtained as exspected in a next step we will sequence our new constructs.
So far fragments were obtained as exspected in a next step we will sequence our new constructs. Overnight cultres for pCDI007 in W3110-RFP and W3110-RFP were inoculated.

15/07/17

Plasmidisolation

We wanted to prepare another SDS Page with pCDI007 in W3110-RFP and to compare this result we also prepared W3110-RFP as a control.
The experiment could not be finished because grow problems ocured.
Another Test Digest of the samples from pCDI005 and pCDI006 with pICD001 as a control was prepared. So far fragments were obtained as exspected in a next step we will sequence our new constructs.

15/07/20

sequencing

The sequenced constructs were not as exspected. The experimental design have to change to get better cloning results.

15/07/21

PCR, overnight culutres

PCR of 15-07-07 was repeated because the Gibson Assembly did not work.
Overnight cultures of CDI003, CDI007, W3110 and W3110-RFP are grown to start a killing assay and an SDS PAGE the next day

15/07/22

SDS-Page, killing assay

Day cultures of CDI003, CDI007, W3110 and W3110-RFP are diluted to an OD600 of 0,1 and induced with 0,5mM IPTG and additional 1% glucose.
Samples of W3110-RFP and CDI007 for SDS PAGE are taken at different times. The amount of taken samples is calculated to 1mL of OD 1,0
For W3110-RFP and W3110 new day cultures are grown to have them at the same OD as the CDI003, CDI007. At an OD of about 0,4 the killing assay was started by mixing CDI003 with CDI007 and CDI003 with W3110. The result at the FACS is that most of the CDI003 cells lost GFP and some cells of CDI007 lost the RFP. So the assay was aborted.
The PCR products of 15-07-21 were put to preperative gel electrophoresis.
The gel extraction was performed with Qiagen gel extraction kit.
Only for fragments 1,2 and 4 sufficient amount of DAN was achieved.
PCR of fragments 3,5,6 and 7 is repeated

15/07/23

Gel extraction, PCR

The PCR products of 15-07-22 were put to preperative gel electrophoresis. Because there are very weak bands for fragments 3 and 7, the PCR is repeated with the original template and DNA from an earlyer PCR for those fragments.
Gel extraction is performed according to the modified gel extraction protocol. Sufficient DNA was achieved for fragments 5 and 6.

15/07/24

plasmidisolation

New plasmid preparation of pICD001 due to strange digestion results on gel.
PCR of fragments 3 and 7 of old and new plasmid DNA

15/07/25

plasmidisolation, CPEC

The PCR fragments of 15-07-24 are used for gel extraction. Gel extraction is performed according to the modified gel extraction protocol.
Sufficient DNA was achieved for fragment 3, for fragment 7 the amount was not completely sufficient but could be used for CPEC anyway.
CPEC:

fragments/volume	fragment 3 (2.78 uL)	fragment 1 (1.29 uL)	fragment 4 (1.50 uL)
ddH2O	4.39 uL
mastermix	40 uL
program	denaturation 98 °C 10 s	annealing 55 °C 30 s	elongation 68 °C 168 s

fragments/volume	fragment 3 (2.78 uL)	fragment 2 (1.16 uL)	fragment 4 (1.50 uL)
ddH2O	4.68 uL
mastermix	40 uL
program	denaturation 98 °C 10 s	annealing 55 °C 30 s	elongation 68 °C 173 s

fragments/volume	fragment 7 (4.39 uL)	fragment 5 (1.8 uL/1:100)
ddH2O	3.814 uL
mastermix	40 uL
program	denaturation 98 °C 10 s	annealing 55 °C 30 s	elongation 68 °C 153 s

fragments/volume	fragment 7 (2.78 uL)	fragment 6 (2.2 uL 1:100)
ddH2O	4.39 uL
mastermix	40 uL
program	denaturation 98 °C 10 s	annealing 55 °C 30 s	elongation 68 °C 158 s

15/07/26

CPEC, analytical gel

CPEC products are run on analytical gel

Bands are visible at about 20kb. Are a little bit higher than expected but that could be due to the cirulized form of the plasmid.
Chemocompetent cells of W3110 are prepared according to protocol "Competent E. coli cells (RbCl)"
CPEC product 1 and 2 and additonally pILS4, pILS5 and pILS6 are transformed to this strain. CPEC product 3 and 4 are tranformed to the hicontrol g10 E. coli strain.
Transformation of CPEC products Volume cells 200µL (100µL of hicontrol g10)
Volume DNA 5µL CPEC products / 1µL pILS4-6
30 min On ice
45s 42°C
2min On ice
Volume LB 800µL (900µL for hicontrol g10)
1h 37°C in shaker
Centrifugation 4min 4000rpm
Discard supernatant Use remaining supernatant for resuspendation
Plate whole volume

15/07/27

transformation

Transformation did not work for all four constructs.
For pILS4, pILS5 and pILS6 many colonys were on the plates.
6 clones each are used to be streaked out on agar plates.
One clone from each construct was grown in an overnight culture to study to growing behaviour compared to the old GFP strain CDI003.
Chemical transformation of the CPEC reactions 1-3 were repeated with chemocompetent top 10 E. coli cells.
In case a new CPEC will have to be started, the PCR for fragment 7 is repeated to get sufficient amount for a new reaction.

15/07/28

transformation, growth experiment

In case the CPEC did not work instead of the transformation the CPEC reaction of 15-07-25 was repeated.
The cultures of the three GFP strains pILS4,5 and 6 are diluted to OD600 of 0,1 and grown over day.
pILS5 (medium promoter) and pILS6 (weakest promoter) grow approximately as W3110. pILS4 (strongest promoter) grows extremely slow comparably to CDI003.

15/07/29

plasmidisolation,digest

Electroporation is done for CPEC reaction 1-3 in NEB TURBO E. coli cells
Miniprep of the 8 pICD006 candidates accrording to thermo scientific miniprep kit. Elution in 30µL Elution Buffer.
Digestion with EcoR1 of the 8 pICD006 candidates and the old and new pICD001

concentration/volume	chemicals
1000 ng	DNA
2 uL	Cut smart buffer
upt to 19 uL total	ddH2O
1 uL	EcoRI

Whole preparation is loaded to a gel

The bands for the pICD006 candidates do not look like they are expected so all samples are discarded.
The old pICD001 looks completely strange but the newly prepared pICD001 looks as expected to this will be used for further experiments.

15/07/30

transformation,digest

Transformation worked for all clones but white and red colonies are visible. Miniprep of the transformant of CPEC reactions 1-3 (pICD003, pICD004, pICD005) according to thermo scientific protocol. Elution in 30µL.
Digestion with EcoR1 (protocol see 15-07-29) and ran on a gel.

15/07/31

sequencing,transformation,digest

pICD003_1, pICD003_2, pICD004_2, pICD004_3, pICD005_1 and pICD005_4 were send to sequencing.
Overnight cultures of pICD006 six different colonies (part of the colonies were plated out on Chloramphenicol Agar plates).
Electroporation is done for CPEC reaction 4 in NEB TURBO E. coli cells.

15/08/04

plasmid isolation, test digest

Miniprep of the transformant of pICD006 according to thermo scientific protocol. Elution in 30µL Growth was obtained on the Chloramphenicol-Agar plates. Test Digest from pICD006 failed caused by running problems from each aliquot.

15/08/05

sequencing

Sequencing results of CPEC constructs:
pICD003-1 and pICD004-2 are correct! Sequencing of pICD005 and 6 clones failed.

15/08/10

plasmidisolation, analytical gel

Miniprep of pICD003-1,pICD004-2, pICD005-7 - pICD005-14 and pICD006-2 - pICD006-9 according to Macherey Nagel Miniprep Kit. Elution in 30µL.
Digestion with EcoR1 of the prepped plasmids (see protocols)

pICD003-1 and pICD004-2 are as expected but the bands for all other samples do not look as expected.

15/08/11

digest, analytical gel, growth experiments

To test if the plasmids are all wrong the digest is repeated with SpH1 (see protocols).

Again no fragments at the desired size so all samples are discarded.
Growth curve of pICD003_1 and pICD004_2 in NEB turbo:
We wanted to check which conditions are needed to express the CDI operon and how growth of NEB turbo is affected by the CDI operon plasmids in different copy numbers (10-12 copys per cell pICD004 and 5-8 copys per cell pICD003). Expression shall be seen during RFP wich is cloned directly behind the CDI operon.
The overnightcultres had an OD-600 of 4.1 for pICD003_1 and 5.2 for pICD004_2
We diluted them to an OD-600 of 0.05 (10 mL) and recorded a growth curve:

strain	0 min	20 min	50 min	87 min	117 min	150 min	180 min	215 min	275 min	335 min
pICD003_1	0.05	0.1	0.18	0.27	0.43	0.74	1.3	1.9	3.1	4.1
pICD004_2	0.05	0.06	0.1	0.13	0.2	0.36	0.7	0.9	2.0	3.2

We induced pICD003_1 after 87 minutes and pICD004_2 after 117 minutes with 0.5 mM IPTG. After one day the aliqouts of pICD003_1 and pICD004 were slightly red. As a result we concluded that NEB turbo dont express proteins properly during log phase. Further information shall be gathered with FACS. So far we can not be sure if only RFP is expressed or RFP and the CDi operon.
Cryo stocks of this two strains (piCD003_1 in NEB turbo [SiGEM 34]and pICD004_2 in NEB turbo [SiGEM 35]) were created (see protocols).

15/08/12

FACS mesurement

Growth curve with NEB Turbo, pICD003, pICD004 (both in NEB Turbo) to take samples for SDS PAGE Cultures are induced with 0,5mM IPTG

	9:20	10:20	11:40	12:20	13:10	15:30	18:00
NEB_Turbo	0.05	0.18	0.59 (induction t=0)	1.32 (t=1)	1.92 (t=2)	4.50 (t=3)	4.90 (t=4)
pICD003	0.05	0.09	0.15	0.33 (induction t=0)	0.56 (t=1)	2.90 (t=2)	4.60 (t=3)
pICD004	0.05	0.12	0.33 (induction t=0)	0.69 (t=1)	1.14 (t=2)	3.30 (t=3)	4.10 (t=4)

Another Growth curve with NEB Turbo, pICD003 in NEB Turbo and pICD004 in NEB Turbo was created this time 0.2 % Glucose was added to the LB broth with Chloramphenicol.
NEB Turbo was here used as a controll. pICD003 in NEB Turbo and pICD004 in NEB Turbo were induced after two hours with 0.5 mM IPTG.

strain	0 min	100 min	125 min
pICD003_1	0.05	0.11	0.14
pICD004_2	0.05	0.16	0.26
NEB_turbo	0.05	0.31	0.56

Before and after induction each sample was tested with FACS. The mesurments was done every hour for seven hours. The last sample was mesured with FACS on 15-08-13.
Result of this FACS mesurements:

strain	t=0	t=1	t=2	t=3	t=4	t=5	t=6	t=7	t=7.2
NEB_turbo	238	262	237	223	183	181	182	227	223
pICD003	14482	13883	10539	7423	4285	3325	5191	off limits	43151
pICD004	7439	14075	7248	2393	1604	1813	2482	off limits	13920

Parameters in Voltage for the first seven mesurements:
FSC: 350, SSC: 385, Blue C 530/30: 350, Yellow Green B 610/20: 650.
Parameters in Voltage for the mesurment 7.2:
FSC: 350, SSC: 385, Blue C 530/30: 350, Yellow Green B 610/20: 500.
The absorption for RFP before induction decreased a bit.
After the induction the absorption for RFP decreased further.
4 hours after the induction the absorption for RFP rises again. 13 hours after the induction the RFP absorption can not be mesured. So the parameters were adjusted and a high level of RFP absorption was obtained.
We concluded that we have leaky expression, but 0.5 mM IPTG leads to a significant rise in the RFP absorption. Also we concluded that after four hours the expression rate of our Proteins (only seen RFP) is higher than the cell division rate. Also the cell division rate could be lower than the expression rate of our Protein.
Transformation of pICD004 and PICD003 into G10 HI high controll strain (protocols).

15/08/13

transformation,SDS-Gels

Transformation of psB4C5 and psB3C5 into G10 HI high control strain (protocols). Plating out of G10 Hi high control stain on LB agar.
Preparing of SDS Gels.

15/08/15

pICD007 with His tag

Construction of pICD008 (pICD007+His Tag):
PCR of whole Plasmid pICD007 using Primers with His Tag and overlap for Gibson assambly (see protocols).
PCR program: 30 cycles like shown below: 38 °C 10 sec
60 °C 15 sec
68 °C 170 sec
after this 30 cycles the PCR product was stored at 4 °C
Analytical Gel

As a next step to recieve only our PCR product we used the enzyme DpnI to destroy the template dna. So we incubated the PCR product at 37 °C for half an hour and denature the restriction enzym afterwards with 80 degree Celsius for 20 minutes.
In the following the PCR product was cleaned up with the Nucleo Spin Gel and PCR Clean up Kit.
In the following we proceeded with a gibson assembly from the PCR product and transformed it into electro competent G10 Hicontrol E.coli cells.

15/08/19

cloning

The transformation of the Gibson assembly was only successful for pICD007 with an additional sequence of six Histidins behind CdiA. So we did the Polymerase chain reaction again for pICD003, pICD004 and pICD007 as a control for the right mastermix.

The PCR was successful for pICD007 and pICD004 (one of two PCR reactions) and was unsuccessful for pICD003. Maybe we change the conditions of this PCR reaction next time.
After this we purificated the PCR product and used Gibson assembly to get a circular plasmids form pICD004 and pICD007 again. The Gibson Assembly was transformed into chemical competent E. coli cells (NEB turbo). Here as a control we transformed pICD007 and the pICD004.
The transformed constructs from Erlangen grew on the Agar plates and single colonys were picked and put into overday cultures ( 3 mL LB broth with CAM and a single picked colony).
The plasmids out of the constructs from Erlangen were isolated with a Plasmid isolation kit and concentrations were mesured.

15/08/20

Erlangen

We got colonys on the plates of pICD007 let them grow and isolated the plasmids.
The constructs from Erlangen were sent to Eurofins for sequencing.

15/08/20

Erlangen

Sequencing results:
pWH403-P5(L9A) with iGEM125fwd: correct sequence!
pWH527-5 and -6 with primers AA537 and AA544: Both clones correct!
BBa_K592008 with iGEM125fwd: Correct sequence!

15/08/21

building new constructs

Preparation of Electrocompetent NEB Turbo according to protocol "Competent E.Coli cells (Electroporation)" starting with 300mL culture.
Transformation of pICD007+His-5 and pICD007+His-6 is done to check the quality of the Electrocompetent cells (Result: Excellent)
Additionally the GA reaction to create pICD004+His is transformed (Result: A lot of colonies. 6 clones are picked and will be sequenced)
PCR to prepare new constructs having an RBS upstream of every CDI gene and with a new promoter because the promoter in the already existing constructs has a point mutation

template	primer	size of the fragment
pICD001,pICD007,pICD003,pICD004	iCD043, iCD045	13kb, 13kb, 15kb, 15kb
pICD007	iCD044,iCD046	1829 bp

Additionally the new promoter K592008 is digested with EcoR1 and Spe1.

For the fragments created with primers iCD043 and iCD045 additional bands are visible. The fragments for pICD007 look fine.
The desired fragments are excised and extracted using the Qiagen Gel extraction kit.
Nanodrop results after gel extraction:
• PCR pICD007 with iCD044 and 046: 26.7ng/µL (lane 4, bottom gel) --> 0.022 pmol/µL

15/08/22

building new constructs

PCR is repeated using the excised DNA

The gel was run too long so the Promoter has run out of the gel. No bands at the desired size for the repeated PCR.
Preparation of Electrocompetent NEB Turbo and G10 Hicontrol according to protocol "Competent E.Coli cells (Electroporation)" starting with 2L culture each.

15/08/23

building new constructs

Digest of K592008 is repeated with doubled volume. And put to preparative gel. The band of K592008 is excised and extracted using the Qiagen Gel extraction kit.

The band of K592008 is excised and extracted using the Qiagen Gel extraction kit. Transformation of pICD007+His-5 is done in NEB Turbo and G10 Hicontrol to check the quality of the Electrocompetent cells prepared on 15-08-22 (Result: Amazing)

15/08/24

plasmid isolation, sequencing

Isolation of the plasmid pICD004 + His-5, and the constructs from Erlangen which were retransformed to get a higher amout of plasmid.
Sequencing of pICD004 + His-5.
Transformation of pICD004 + His-5 into G10 Hicontrol. We want to check in the next days if we can prove the presence of our CdiA protein with western blotting.
Another Digest of K592008 to get a higher amout of the d.n.a sequence which codes for the new promoter we want to use for CdiA.
Overnight cultures for pICD007 + His and G10 Hicontrol for the SDS page and western blot tomorrow. Repeated PCR of pICD004 and 007 with primer ICD043 and 045 (see 15-08-21) Fragment of the desired size is excised and extracted using the "modified gel extraction protocol"

15/08/25

Western Blot, sequencing

Sequencing results:
• PICD004-His with primer iGEM160-rev: Clone 3 is correct, clone 1 has a point mutation destroying the CdiI start codon --> Clone 3 will be used and becomes pICD012
• pWH1413 with AA537 and AA544: tetR sequence correct!
• pWH527 with AA537 and AA544: tetR sequence correct!
• pICD007-His with primer iGEM160-rev: Both clones, 5 and 6, are correct! --> Clone 5 becomes pICD013
Excised bands from yesterday's PCRs --> gel extraction (Zymo kit, which is more suitable for long fragments), eluted in 15 ul • PICD004 PCR: 85.5 ng/ul, fragment length 14767bp --> 0.009 pmol/µl
• PICD007 PCR: 73.1 ng/ul, fragment length 12897 bp --> 0.009 pmol/µL
With pICD0013 we want to do an Western Blot with Histidin (6x) monoclonal antibodys. So we prepared dayculutres of pICD0013 and G10 Hicontrol as a negative control. We dont have a positive control.
Absorbance at 600 nm for the overnight culutres was: pICD0013 = 2.7 and G10 Hicontrol = 3.6
Both culutures were diluted to an OD = 0.05 in a 200 mL culture. LB Broth without additional glucose was used. At an OD of 0.2 for pICD0013 and an OD of 0.43 for G10 Hicontrol both were induced with 0.5 mM IPTG. An aliquot of equivalent 1 mL of cells at OD-600 = 1.0 was collected and spined down at maximum speed for one minute. We removed the supernatant and store the cells as t = 0 at -20 degree Celsius. This t = 0 was collected after 100 minutes of growth.
The let both culutures grow for 8 hours at 37 °C degree celsius and 220 rpm. After this 8 hours of induced cell growth we harvested both culutres. The OD-600 mesured value of pICD0013 was 3.0 and of G10 Hicontrol was 4.2. From both culutres an aliquot of equivalent 1 mL of cells at OD-600 = 1.0 was collected and spined down at maximum speed for one minute. We removed the supernatant and store the cells as t = 0 at -20 degree Celsius. This sample was named t = 1.
t = 0 and t =1 were resuspend each in 65 µL of 1 x SDS-Page sample buffer ( 900 µL 4 x SDS sample buffer + 100 µL Beta-Mercaptoethanol).
We also wanted to test for solubility:
So we pellet 200 mL of each culture and resuspended in 2 mL Phosphat Buffer and added 0.1 mg/µL Lysozym. After the incubation time of half an hour at 37 degree celsius we lyse the cells with sonication ( Amplitude 90 %, 1 s pulse, 2 x 10 minutes). Soluable and Insoluable were seperated by spinning down at 10000 g at 4 °C for 20 minutes. 20 µL of Supernatant were added to 20 µL 1x SDS Page Buffer (Solubale fraction). The pellet was resuspended in Phosphat buffer pH 7.2 and 20 µL of this were added to 20 µL of 1x SDS Page Buffer (insoluable fraction).
SDS Gel and Western Blot were continued next day.

15/08/26

Western Blot, sequencing

Gel extraction of the new Promoter K592008 does not work neither with Qiagen Gel extraction kit nor following the "Modified gel extraction protocol"
So Gibson assembly is done with unpurified and just digested plasmid in hope that only the correct fragment will be able to assemble.
PCR of 15-08-21 for the CDIB fragment from backbone pICD007 and primers iCD044 and iCD066 and purified using the Thermo scientific PCR clean up kit.
Gibson is performed using one of the backbones pICD004 and pICD007 each of them containing the CDI operon without CDIB, promoter and CDIB.
The two GA reactions are transformed into G10 Hicontrol electrocompetent cells.
Running an SDS Page for Western Blot:
Gel: pICD0013 was used on the lanes2-5 and G10 Hicontrol as a negative control on the lanes 7-10 Lanes: 1 Ladder; 2 cell lysat before induction; 3 cell lysat after 8 hours of induction, 4 supernatant, 5 insoluable part, 6 ladder, 7 cell lysat before induction; 8 cell lysat after 8 hours of induction, 9 supernatant, 10 insoluable part
We prepared the Western Blot after a standart protocol.

15/08/27

transformation, failed western blot

Colonies of transforemed cells are picked to do miniprep the following day.
We use a monoclonal antibody to mark His-6 tags. But no signal could be obtained. Western Blot failed!

15/08/28

Killing assay, transformation, failed western blot

The picked candidates of the GA are used for miniprep using the Macherey Nagel miniprep kit.
The plasmids are digested with Sac1 and put to analytical gel.
The fragments have a size equal to the size of digested plasmid carrying the promoter K592008 so the GA did not work and undigested backbone was transformed.
Killing Assay
Overnight cultures of G10 hicontrol, pSB4C5, pICD004, pILS5 were prepared on 15-08-27.
OD was measured of the overnight cultures and during the day
Induction at OD of about 0,2 with 0,5mM IPTG

strain	overnight culture	9:30	10:30	11:05	12:40	13:50	14:50	16:10
G10 uninduced	3.6	0.05	0.12	0.23 (induction at 11:20)	0.67		0.79	1.04
G10 induced					0.61		0.81	1.00
pICD004 uninduced	1.8	0.05	0.14	0.23 (induction at 11:20)	0.46		0.84	1.14
pICD004 induced					0.47		0.84	1.10
pSB4C5 uninduced	2.7	0.05	0.06	0.09	0.09	0.19 (induction at 13:50)	0.27
pSB4C5 induced							0.26
pILS5 uninduced	3.5	0.05	0.12	0.24 (induction at 11:20)	0.46		0.8	1.04
pILS5 induced					0.47		0.83	0.98

As pILS5 and pICD004 grow very similar, the growth in coculture should be similar as well to see an effect of growth inhibition.
Cultures are grown in an incubator with 37°C and 200rpm until induction. After induction the flasks were put in an incubator without shaking. The flasks are shaked every 30 minutes by hand. Cultures of pICD004 and pILS5 are mixed directly after induction in a ratio of 1:1 and 10:1 (10*piCD004: 1*pILS5) induced as well as uninduced (as control)

well	1	2	3	4	5	6	7	8	9	10	11	12
sample	G10 uninduced	G10 induced	pICD004 uninduced	pICD004 induced	pILS5 uninduced	pILS5 induced	pSB4C5-RFP uninduced	pSB4C5-RFP induced	pICD004+pILS5 1:1 uninduced	pICD004+pILS5 10:1 uninduced	pICD004+pILS5 1:1 induced	pICD004+pILS5 10:1 induced

Samples are measured in the FACS, directly after induction (t0, 1:4 dilution), after 2h (t1, 1:10 dilution), after 3h (t2, 1:20 dilution), after 4h (t3, 1:20 dilution) and on the folloPCRwing day (t4, 1:20 dilution).
results: wait for our awesome presentation.

15/08/29

Gibson assembly, PCR, sequencing

Gibson assembly is done to clone synthesized CDIA-CT into pICD004
PCR with primers ICD047 and ICD016 to amplify the whole plasmid except the CT After PCR purification 29,5ng/µL
Sequenced CTs are used 3 fold molar.

15/08/30

Overnight culutre

Colonies on all plates. 6 clones each are picked and grown over night to do miniprep the following day.

15/08/31

plasmid isolation, PCR, digest

Miniprep of all clones using the macherey nagel kit. Elution in 30µL elution buffer.
Test PCR to test if old or new CT is in the plasmid.
digest:
iCD031 and 160 rev would give a band if new CT is in the plasmid.
iCD032 and 160 rev would give a band if new CT is in the plasmid
Bands visible for pICD009-2, pICD010-4, pICD010-6, pICD011-6.
Sent to sequencing using primers iCD031 and 160 rev.
More clones are picked for pICD008, pICD009 and pICD010 in case that sequencing results are wrong.

15/09/01

sequencing, overnight culture

Sequencing results: PICD009-2 with primers ICD031 and 160rev: Entire sequence correct! --> becomes pICD009
PICD010-4 with same primers as above: Missing A in CdiA gene --> frameshift!
PICD010-6 with same primers as above: GTG to GTC in base pair position 8340 of CdiA --> Silent mutation. We decided to continue with this plasmid --> becomes pICD010
PICD011-6 with same primers as above: TTC to TCC in CdiA gene! Results in F to S mutation. Cannot be used!
New minipreps of pICD008 and pICD011 candidates are prepared.

15/09/02

sequencing

Sequencing results:
PICD008-12 with primers ICD031 and 160rev: Sequence correct! --> becomes pICD008
PICD011-10 with same primers as above: point mutation, will not be used
PICD011-7 with same primers as above: All correct! --> becomes pICD011
PCR to create fragments for two new target strains?

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