Revision as of 19:47, 24 October 2010

Results

Photosensor

Biobrick assembly and sequence

We succesfully assembled the biobrick 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

Stability assay

Stability assay.

0 generations	Dilution number	Colonies counted on plate	Cfu	PS-chlor/PS+chlor
PS1 + chlor	6	168	8.4E+09	100
PS1 - chlor	6	157	7.85E+09
PS2 + chlor	6	192	9.6E+09	100
PS2 - chlor	6	216	1.08E+10
20 generations
PS1 + chlor	6	157	7.85E+09	59.24528302
PS1 - chlor	6	265	1.325E+10
PS2 + chlor	6	130	6.5E+09	63.41463415
PS2 - chlor	6	205	1.025E+10
40 generations
PS1 + chlor	6	143	7.15E+09	95.97315436
PS1 - chlor	6	149	7.45E+09
PS2 + chlor	6	168	8.4E+09	92.81767956
PS2 - chlor	6	181	9.05E+09
60 generations
PS1 + chlor	6	147	7.35E+09	89.63414634
PS1 - chlor	6	164	8.2E+09
PS2 + chlor	6	134	6.7E+09	104.6875
PS2 - chlor	6	128	6.4E+09
80 generations
PS1 + chlor	6	127	6.35E+09	136.5591398
PS1 - chlor	6	93	4.65E+09
PS2 + chlor	6	99	4.95E+09	104.2105263
PS2 - chlor	6	95	4.75E+09
100 generations
PS1 + chlor	5	130	650000000	125
PS1 - chlor	5	104	520000000
PS2 + chlor	5	124	620000000	118.0952381
PS2 - chlor	5	105	525000000

Number of generations	PS1	PS2	PS (average)	Standard deviations
0	100	100	100	0
20	59.24528	63.41463	61.32995858	2.948176455
40	95.97315	92.81768	94.39541696	2.231257632
60	89.63415	104.6875	97.16082317	10.64432845
80	136.5591	104.2105	120.3848331	22.87392395
100	125	118.0952	121.547619	4.882403965

Retinal

Biobrick assembly and sequence

We succesfully assembled the biobrick 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

Stability assay

Flagella

Biobrick assembly and sequence

We succesfully assembled the biobrick 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!

@@ Line 469: / Line 469: @@
 === Stability assay ===
-===Motility assay===
-<br>Exp. 1<br>
-The purpose of this experiment is to see if the cells containing pSB1C3-K343004 move farther than the wild type (MG1655) and the negative control (DH5alpha). This would be an indication of hyperflagellation. We also want to test if it makes a difference in the motility wether the bacteria contain low-medium- or high-copy plasmids. <br><br>
-For the motility experiments we added 5ul of an ON culture to petridishes containing motility agar (LB media with 0.3% agar) instead of regular LA (Luria agar). This semi-solid media lets the bacteria swim more easily. <br>
-The plates were incubated at 37 degrees celcius for 24 hours.<br><br>
-[[Image:Team-SDU-Denmark-Flagellamotility-exp1-DH5a.JPG|250px|DH5alpha]]
-[[Image:Team-SDU-Denmark-Flagellamotility-exp1-MG1655.JPG|250px|MG1655]]<br><br>
-[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB1C3_(LA+chlor).JPG|250px|pSB1C3-FlhDCmutCP(LA+chlor).]]
-[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB3K3_(LA+kan).JPG|250px|pSB3K3-FlhDCmutCP(LA+Kan)]]
-<br><br>
-The upper two plates did not contain antibiotics, and therefore contamination colonies are seen.<br>
-The upper left picture is of ''E. coli'' strain '''DH5alpha''' that does not express flagella and therefor movement in the media should, as seen on the picture, be minimal. The upper right picture is of the wild type ''E. coli'' strain '''MG1655''', this strain has about 8-10 flagella per cell. These cells are, as seen, expected to move farther than the DH5alpha but not as far as the transformed cells. The lower left picture is of ''E. Coli'' strain '''MG1655 with pSB1C3-K343004''' this shows that these bacteria move farther than the wild type and the negative control. The lover right picture is of ''E. coli'' strain '''MG1655 with pSB3K3-K343004''' these bacteria move farther than the wild type, the negative control and MG1655 with pSB3K3-K343004.<br><br>
-From the pictures above we can definately se that the bacteria containing our part is much more motile than the wild type. We assume this is caused by overexpression of the FlhDC master flagella operon which leads to hyperflagellation of the cells. <br> The two buttom pictures show that bacteria with pSB1C3-K343004 have not moved as far as the bacteria containing pSB3K3-K343004. pSB1C3 is a high copy plasmid while pSB3K3 is a low-medium copy plasmid. The promoters in K343004 is a constitutive promoter (tetR repressable promoter). Bacteria containing a high copy plasmid with a constitutive promoter are more metabolically challanged than bacteria containing a low- or medium-copy plasmid with a constitutive promoter because of the higher number of plasmids per the cell. Therefore the high copy plasmid bacteria are less motile than low- or medium-copy plasmid bacteria.<br>
 == OD measurment of MG1655 ==

Team:SDU-Denmark/project-r

From 2010.igem.org

Revision as of 19:47, 24 October 2010

Results

Photosensor

Biobrick assembly and sequence

Effect

Stability assay

Retinal

Biobrick assembly and sequence

Effect

Stability assay

Flagella

Biobrick assembly and sequence

Effect

Stability assay

OD measurment of MG1655

Contents