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

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