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Team:SDU-Denmark/project-r
From 2010.igem.org
Results
Photosensor
Biobrick assembly and sequence
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343007 K343007], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343003 and the terminators B0010 and B0012.
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.
Sequencing results (as .ab1 files):
Sequencing results K343007
When comparing the sequence that came back from sequencing with the theoretical sequence the part should have it is identical, except for a silent mutation in the coding sequence, which does not change the amino acid sequence.
Effect
Our results from the experiments with semi-solid agar confirm that the biobrick does indeed couple itself to the bacterial chemotaxis pathway and modify the bacterial motility pattern by modifying the tumbling frequency. For further information on this conclusion see Characterization - K343007 - 1. Growth of bacterial culture on semi-solid agar plates (Experiment 1 and 2)
The videomicroscopy indicates that blue light with a wavelength around 500nm leads to CheA's autophosphorylation being downregulated. This means that the bacterial tumbling frequency gets reduced and the bacteria will spend an increased time in the "run" mode of propulsion. This means that the bacteria will travel further in a certain amount of time, compared to wildtype bacteria. More details can be found here Characterization - K343007 - 2. Videomicroscopy and computer analysis of bacterial motility
Regarding the stability of the plasmid, we carried out a stability assay according to protocol [1] There was no observeable difference between the cultures plated out on plates with and without antibiotics. From this we conclude that the plasmid does not significantly increase the stress on the bacteria, since they don't seem to lose the plasmid after 5 days. Since this did not happen the plasmid seems to be stable. For a closer look at the results look here:
Retinal
Biobrick assembly and sequence
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343006 K343006], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343002 and the terminators B0010 and B0012.
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.
Sequencing results (as .ab1 files):
Sequencing results for K343006
When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
Effect
Flagella
Biobrick assembly and sequence
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343004 K343004], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343000 and the terminators B0010 and B0012.
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.
Sequencing results (as .ab1 files):
Sequencing results for K343004
When comparing the theoretical sequence with the sequence done on the part K343004 the two sequences was identical.
Effect
Stability assay
OD measurment of MG1655
| Mg1655 | PS in pSB3T5 | PS in pSB1C3 | flhDC in pSB3K3 | flhDC in pSB1C3 | NinaB in pSB1C3 | |||||||
| Hours | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 |
| 0 | 0.024 | 0.026 | 0.028 | 0.03 | 0.024 | 0.024 | 0.028 | 0.027 | 0.028 | 0.027 | 0.027 | 0.027 |
| 1 | 0.082 | 0.03 | 0.058 | 0.06 | 0.059 | 0.063 | 0.069 | 0.077 | 0.052 | 0.051 | 0.084 | 0.072 |
| 2.5 | 0.58 | 0.65 | 0.42 | 0.45 | 0.48 | 0.44 | 0.52 | 0.6 | 0.38 | 0.34 | 0.31 | 0.54 |
| 3 | 1.14 | 1.16 | 0.99 | 0.91 | 0.94 | 0.94 | 1.06 | 1.16 | 0.84 | 0.78 | 1.04 | 1.03 |
| 3.83 | 1.54 | 1.66 | 1.52 | 1.31 | 1.38 | 1.37 | 1.54 | 1.72 | 1.33 | 1.25 | 1.91 | 1.45 |
| 5 | 4.4 | 4.3 | 3.2 | 3.4 | 4.2 | 3.3 | 4.2 | 2.2 | 3.9 | 4 | 3.9 | 3.9 |
| 6 | 4.7 | 4.7 | 2.8 | 4.3 | 4.2 | 3.1 | 4.7 | 4.4 | 3.8 | 5 | 3.4 | 4.7 |
| 7 | 4 | 3.8 | 3.3 | 3.8 | 4.2 | 3.2 | 4.3 | 4.7 | 4.9 | 4.5 | 3.7 | 3.9 |
| 8 | 5.5 | 6.1 | 3.9 | 4 | 3.5 | 6.8 | 4.2 | 4.8 | 5.4 | 5.3 | 4.6 | 4.6 |
| 9 | 4.1 | 4.1 | 3.4 | 2.6 | 3.6 | 4.7 | 2.6 | 3.1 | 3.7 | 3.1 | 2.6 | 3.6 |
| 10 | 4.6 | 6.1 | 5 | 4.8 | 7.8 | 4.5 | 5 | 6 | 5.3 | 5.3 | 4.4 | 4.7 |
| 11 | 5.1 | 6.2 | 4.7 | 4 | 6.1 | 7.1 | 6.6 | 8.3 | 6.6 | 6.4 | 6.2 | 4.1 |
| 12 | 5.1 | 5 | 3 | 4.4 | 4.4 | 3.9 | 4 | 4.8 | 5 | 5.3 | 4 | 4.7 |
| 24 | 5.2 | 5.6 | 3.3 | 5.4 | 6.4 | 5.8 | 4.6 | 6.4 | 7.1 | 7.4 | 5.6 | 5.2 |
This is where it gets really interesting. Just look what we've made!
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