Team:UCopenhagen/Notebook
Lab notebook
The notebook is a chronological overview of what has happened in the lab. It is organized after each experiment.Cloning
Making BioBricks
Liposome characterization
Onion characterization
Leakiness characterization
Membranes and microscope
Week 28
Learning to make liposomes – 12/07/18
We were introduced to the process of making liposomes by the nice people in Nikos Hatzakis lab, Søren Schmidt-Rasmussen Nielsen and Mette Galsgaard Malle who gave us the Preparing liposomes protocol. We made some tryout liposomes, that were flashed freezed and stored in -20 oC.
Selma, Sofia and AttilaWeek 34
Transforming SIEC strains with GFP plasmids – 24/08/18
To visualize our bacteria in the microscope together with a membrane we needed it transformed with a GFP plasmid with constitutive expression. We used plasmids from the distribution kit that functioned as Test Device 1 (BBa_J36400) and positive control (BBa_I20270) in the InterLab study. Transformation as described in Transformation protocol until step 14, the following combinations of strain and plasmid was made:
- SIEC x BBa_J36400
- SIEC x BBa_I20270
- SIECΔp1 x BBa_J36400
- SIECΔp1 x BBa_I20270
Plates were made from agar and LB with chloramphenicol (34 μg/mL) (CAM). Plates with bacteria spent 17 hours in the incubator before being kept in fridge. Conclusion: hereafter we used the bacteria transformed with BBa_I20270.
SelmaWeek 35
Testing transformed bacteria for fluorescence – 27/08/18
To test if the transformation was successful fluorescence were measured on a plate reader. Colonies from transformation 24/08/18 was picked into 50 mL falcon tubes with 5 mL LB media that also contained 1% arabinose (not necessary). Incubated at 37 oC at 130 rpm for 6 hours. Triplicates of each 100 μL of culture was pipetted into wells of a 96 well-plate. Fluorescence and absorbance was measured. RESULTS, distribution AND SETTINGS Glycerol stocks were made. Conclusion: because a negative control was forgotten, results were inconclusive.
Selma Co-transformations of SIEC strains 1 – 28/08/18 We made co-transformed bacteria for the membrane experiments that needed bacteria with both a reporter to visualize the bacteria and and the T3SS signal tagged reporter. We used constitutive active GFP, BBa_I20260, in a plasmid with a kanamycin resistance gene found in the distribution kit plate 4 well 18A. For our signal tagged reporter, we used mCherry plasmids made the xx/xx/18 by Nat. Transformation as described in Transformation protocol until step 14, the following combinations of strain and plasmid was made:- T1: SIEC x plasmid plate 4: 18:A x ss-mCherry (repeats a and b)
- T2: SIECΔp1 x plasmid plate 4: 18:A x ss-mCherry (repeats a and b)
- T3: SIEC x plasmid plate 4: 18:A x Chaperone-ss-mCherry
- T4: SIECΔp1 x plasmid plate 4: 18:A x Chaperone-ss-mCherry
- T5: Mach1 x plasmid plate 4: 18:A
For the co-transformations plates were made with LB and agar with both kanamycin (50 μg/mL) (KAN) and CAM. Before plating, the bacteria was spinned down, 800 μL supernatant was discarded and bacteria resuspended. Mach1 with plasmid plate 4: 18:A were plated on CAM plates for backup of the plasmid. Plates with bacteria spent 15 hours in incubator. Colony count:
|
Transformation |
Colonies |
|
T1,a |
3 |
|
T1,b |
0 |
|
T2,a |
1 |
|
T2,b |
4 |
|
T3 |
0 |
|
T4 |
0 |
Conclusion: only co-transformations with plasmids without chaperones gave colonies.
Selma