Team:Marburg/Labbook/CDI

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
ILS
Figure 1: Ladder, PCR 1, PCR 2

Ran of analytical gel. No bands were observed.

15/05/19

PCR

ILS
Figure 1: Ladder, PCR 1, PCR 2

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)

ILS
Figure 1: Ladder, - , PCR
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
ILS
Figure 1: Ladder,#1, #2, #3, #4, #5, #6, #7, #8, #9, #10, #11, #12, Ladder
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"
ansformation: 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

Transformation into chem. comp. cells

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

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