Difference between revisions of "Team:Marburg/Labbook/CDI"
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Sascha Grobe (Talk | contribs) |
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Made LB plates with CAM and 0.8 mM IPTG | Made LB plates with CAM and 0.8 mM IPTG | ||
| + | |||
| + | <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;"> | ||
| + | <tr style="background-color:#f66300;"> | ||
| + | <td>strain</td> | ||
| + | <td> LB media </td> | ||
| + | <td> TB media</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>W3110-WT</td> | ||
| + | <td class='col'>6.6</td> | ||
| + | <td class='col'>3.1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>W3110-RFP</td> | ||
| + | <td class='col'>5.8</td> | ||
| + | <td class='col'>2.8</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>CDI002</td> | ||
| + | <td class='col'>4.1</td> | ||
| + | <td class='col'>1.9</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>CDI003</td> | ||
| + | <td class='col'>4.1</td> | ||
| + | <td class='col'>1.4</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | -> Strains grow roughly twice as dense in LB than in TB <br> | ||
| + | -> Strains harboring a high copy plasmid (CDI002 and 003) grow only half to 2/3rd as dense as strains without plasmid | ||
| + | <br> | ||
| + | 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. | ||
| + | |||
| + | |||
| + | |||
| + | <h1>15/06/11</h1> | ||
| + | |||
| + | <h2>swarming assay; Transformation</h2> | ||
| + | <p> | ||
| + | |||
| + | 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: <br> | ||
| + | Transformation of non-competent E.coli cells <br> | ||
| + | To see how much Plasmid we need to get colonies on LB+CAM plates we used different amounts of Plasmid for the Transformation Protocol. <br> | ||
| + | 1. 0.1 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002) <br> | ||
| + | 2. 1 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002) <br> | ||
| + | 3. 10 µL Plasmid pCDI001 without CdiA-CT and CdiI (pICD002) <br> | ||
| + | |||
| + | <h1>15/06/12</h1> | ||
| + | |||
| + | <h2>swarming assay; Transformation</h2> | ||
| + | <p> | ||
| + | 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. <br> | ||
| + | |||
| + | Swarming assay <br> | ||
| + | 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. <br> | ||
| + | After 3 days incubation at 30°C the plates were put on a blue light screen to visualise fluorescence.<br> | ||
| + | <figure style="text-align:center;"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/2/2d/MR_pic_CDI_12.06.1.jpg" /> | ||
| + | <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"> | ||
| + | <b>Figure 1:</b> | ||
| + | ladder, pcr product 1 | ||
| + | |||
| + | </figcaption> | ||
| + | </figure> | ||
Revision as of 01:48, 13 September 2015
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)
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 |
| temperature | time |
| 50 °C | 15 min |
| 16 °C | store |
• 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 |
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 |
| temperature | time |
| 50 °C | 15 min |
| 16 °C | store |
• 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 |
| Digest | volume |
| DNA template | 10 uL |
| Cutsmart buffer | 2 uL |
| Distilled water | 7 uL |
| SacI | 1 uL |
| total | 20 uL |
Analytic gel ; correct size abt 6700 and 8000
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
• 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 |
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 |
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.
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 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.
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