Memory Units
cI Autoinducing Loops
The autoinducing loop driven by cI will function as our first memory unit.
We aimed to find a combination of RBS, transcription factor and degradation tag that enables us to toggle between the two steady states in a robust way. To estimate the rate in which single cells are activated by too much leakage of cI protein we measured the fluorescence in uninduced cells with a flow cytometer. To measure the activation we used a construct with the PRM promoter producing GFP with an LVA tag to make dynamic measurement possible.
| cI autoinducing loop 1 (BBa_K00) after 4 hours of incubation
|
Figure 1. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
|
|
| Number
| % of total
| Mean fluorescence
|
| Visible
| 50000
| 100
| ---
|
| Fluorescent | 9180 | 18,36 | 11419
|
| Non-Fluorescent | 34424 | 68 | 5974
|
|
| Two populations are visible on the dotplot. We seperated both populations on the histogram to show that the major peak of nonfluorescent cells overlaps the minor peak of fluorescenting cells.
|
| cI autoinducing loop 6 (BBa_K00) after 4 hours of incubation
|
Figure 2. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
|
|
| Number
| % of total
| Mean fluorescence
|
| Visible
| 50000
| 100
| ---
|
| Fluorescent | 9180 | 18,36 | 11419
|
| Non-Fluorescent | 34424 | 68 | 5974
|
|
| Only one population is visible on the dotplot. The histogram was obtained in the same fashion as in figure 1 to show that there are cultures with activated cI loops and cultures without.
|
These measurements indicate activation and by this bimodal behaviour of cells containig BBa_. But no activation of BBa_ and several other constructs.
In these measurements we used GFP-LVA. The degradation seems to be about as fast as the production so only faint signals are visible. We decided to repeat the measurement with similar constructs but with GFP instead of GFP-LVA as an output.
| | no-tag | DAS | LVA
|
| RBS 0,07 |
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607019 BBa_K607019]
|
|
Ci12ui2.png
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607014 BBa_K607014]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607010 BBa_K607010]
|
|
| RBS 0,3 |
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607020 BBa_K607020]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607016 BBa_K607016]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607011 BBa_K607011]
|
|
| RBS 0,6 |
Ci3ui2.png
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607021 BBa_K607021]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607017 BBa_K607017]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607012 BBa_K607012]
|
|
| RBS 1 |
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607022 BBa_K607022]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607018 BBa_K607018]
|
|
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607013 BBa_K607013]
|
|
LasR Autoinducing Loops
Our second memory unit consists of a LasR gene(BBa_) driven the LasB promoter(BBa_). LasR only activates transcription in the presence of HSL, this was added to the medium in a concentration of 100nM.
| cI autoinducing loop 1 (BBa_K00) after 4 hours of incubation
|
|
Figure 1. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
|
|
| Number
| % of total
| Mean fluorescence
|
| Visible
| 50000
| 100
| ---
|
| Fluorescent | 9180 | 18,36 | 11419
|
| Non-Fluorescent | 34424 | 68 | 5974
|
|
| Two populations are visible on the dotplot. We seperated both populations on the histogram to show that the major peak of nonfluorescent cells overlaps the minor peak of fluorescenting cells.
|
| cI autoinducing loop 6 (BBa_K00) after 4 hours of incubation
|
|
Figure 2. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
|
|
| Number
| % of total
| Mean fluorescence
|
| Visible
| 50000
| 100
| ---
|
| Fluorescent | 9180 | 18,36 | 11419
|
| Non-Fluorescent | 34424 | 68 | 5974
|
|
| Only one population is visible on the dotplot. The histogram was obtained in the same fashion as in figure 1 to show that there are cultures with activated cI loops and cultures without.
|
"
