Team:Gdansk-UG/Notebook

Week 1
6 flasks were prepared with different alcohol concentration for determining M.organophilum tolerance to methanol, tolerance of aerobic conditions and tolerance to ethanol.
Medium used for cultivating M.organophilum contains:

• 5g peptone
• 3g meat extract
• Distilled water to 1l
• pH adjusted to 7.0
• 

Week 2
Inoculating liquid medium with E.coli TOP10 F’ (in preparation for transformation).
OD600  of E.coli after one night – 0,718
Preparing competent E.coli TOP10 F’ cells.
Transformation of E.coli with iGEM Transformation efficiency Kit according to protocol provided by iGEM - http://parts.igem.org/Help:Transformation_Efficiency_Kit.
Transformation of E.coli TOP10 F’ cells with BioBricks:

• BBa_K316003 – 3C in plate 1
• BBa_K823017  - 3D in plate 1
• BBa_K895006  - 15L in plate 1

Incubation in 37° C.
Transformation results:

Inoculation of plates with basic medium and 25mg/l chloramphenicol with colonies from 3C and 15L plate.
Week 3
Inoculation liquid medium with cholamphenicol (25mg/l) with 1 colony of transformed E.coli (3C and 15L).
 Inoculating liquid medium with E.coli TOP10 F’ strain to prepare next batch of competent cells.
Preparing competent cells.
Isolation of plasmid DNA from transformed E.coli (3C and 15L).
OD600  of cell suspension (inoculated on 30.07.13).

Week 4
Preparing M. organophilum cells to isolation of genomic DNA.
Isolation of genomic DNA of M.organophilum.
Measuring concentration of isolated DNA with NanoDrop – result not good enough for performing PCR.
Inoculation of medium with M.organophilum for next isolation.
Isolation of genomic DNA from M.organophilum with cold lysis buffer.
Results measured with NanoDrop.

Week 5
Running PCR with previously isolated genomic DNA.
Running agarose electrophoresis in search of PCR product.

In wells 5, 6 and 7 we can see a PCR product that we were aiming to have.
Isolation of PCR product from gel with DNA purification kit. Measuring results with NanoDrop – results were not good enough. Purification of PCR product from PCR reaction mix. Measuring results with NanoDrop – results were not good enough.
Week 6
Isolation of genomic DNA from M.organophilum.
Running PRC with temperature gradient from isolated DNA.

Running gel electrophoresis with PCR reaction mix. Purification of PCR product from gel.
Results:

Week 7
Linear backbone pSB1C3 digestion according to protocol provided by iGEM.
Ligation of backbone with PCR product from well 10 – failed.
Ligation with restriction enzymes from a different company – failed.
Week 8
Ligation with restriction enzymes from yet another company – successful.
Transformation of competent cells – failed.
Transformation of competent cells with another batch of competent cells – successful.
Week 9
Isolation of plasmid DNA.
Running agarose electrophoresis to check if the plasmid contains insert – it does.
Sending a BioBrick part to the registry.
Week 10
PCR: promoter with restriction sites suitable for cloning into pKT230 plasmid – successful.
Ligation of PCR product with pKT230 plasmid – successful.

Protocols.

Chemical transformation.
Perform the same procedure for control test.

1. Remove tube of frozen competent cells from -70 C and place on ice.
2. Remove 100ul per transformation into a sterile pre-chilled Eppendorf tube.
3. Add 25ng of DNA (2-10ul) to the tube. Flick the tube several times.
4. Leave the tube on ice for at least 10 minutes. 
5. Heat shock the cells for 45s at 42 C.
6. Place tubes on ice for 2 minutes. 
7. Add 900ul of preheated to 37°C LB medium and incubate for 1 hour at 37 C with shaking at 225 rpm.
8.  Plate 100ul of the cells onto antibiotic plates.
9.  Place plates in the 37°C incubator and grow overnight.

Preparation of competent cells.
1. Prepare 25ml of sterile 50mM CaCl2 and cool it in a refrigerator.
 2. Innoculate one 1ml of LB medium with appropriate strain of E.coli.
3. Culture it overnight in 37°C.
4. Innoculate 40 ml of medium with overnight culture and culture it in 37°C.
 5. When OD600 = 0,2 – 0,25 cool the culture on ice for 15 minutes.
6. Centrifuge (5 000 rpm, 10 min, 4 °C).
7. Suspend the pellet in 20ml of cold CaCl2.
8. Incubate on ice for 20 minutes.
9. Centrifuge (5 000 rpm, 10 min, 4 °C) .
10. Suspend the pellet in 1ml of cold CaCl2.
11. Incubate for 30-40 minutes on ice.

Plasmid DNA isolation – according to a protocol delivered with a kit (GenElute™ Plasmid Miniprep Kit).

Genomic DNA isolation – according to a protocol delivered with a kit (Wizard® Genomic DNA Purification Kit).

Purification of DNA from agarose gel and PCR mixture – according to a protocol delivered with a kit (Wizard® SV Gel and PCR Clean-Up System).

Agarose electrophoresis – 2% gel.

1. Dissolve 1,2 agarose in 60 ml of 0,5xTBE buffer. Heat the mixture up.

 2. When cooled to about 60°C, pour the mixture into the gel mold. Wait for it to cool down.

3. Mix the DNA sample with GelRed dye and loading buffer. Load the sample and DNA ladder.

4. Run the electrophoresis in 0,5xTBA, for 30 min, under 60V.

PCR program for promoter with BioBrick overhangs.

Gradient of temperatures in different PCR tubes (2 bolded values in the previous table):

PCR mix:
12,5 ul of MasterMix (GoTaq® G2 Hot Start Master Mix)
0,5 ul of 1uM forward primer
0,5 ul of 1uM reverse primer
1 ul of 138ng/ul genomic DNA
10,5 ul MiliQ water