Part:BBa_K1022114
pT7 : Lysis Device
This bio-brick codes for the promoter pT7(BBa_I712074) followed by the lysis device(BBa_K112808).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1404
Illegal NheI site found at 1427 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1009
Illegal AgeI site found at 1079 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1660
Characterization
For more info, visit [http://2013.igem.org/Team:TU-Delft/Killswitch TU Delft iGEM13 Wiki]
Lysis Experiment
Introduction:
The construct BBa_K1022114 is made by ligating the pT7 promoter BBa_I712074 in front of the lysis device from the biobrick BBa_K112808. This is to analyze how the lysis device can be controlled if needed.
Description:
The E. coli cells transformed with pT7 lysis cassette (BBa_K1022114) is grown on a plate reader which is capable of shaking and heating to 37˚C to take readings of the cells in exponential phase at every 10 minutes. Different range of IPTG concentration is used to characterize the bio – brick BBa_K1022114. At time point, 160 minutes, IPTG is added according to the table below.
| IPTG concentration | No cells + 1mM | 0.1mM | 0.2m | 0.3mM | 0.4mM | 0.5mM | 0.6mM | 0.7mM | 0.8mM | 0.9mM | 1mM | Cell + No IPTG |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| LB(µL) | 90 | 94 | 93 | 92 | 91 | 90 | 89 | 88 | 87 | 86 | 85 | 95 |
| Cells(µL) | - | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| 10X IPTG(µL) | 10 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | - |
For a detailed set up of the experiment see [http://2013.igem.org/Team:TU-Delft/Protocol_12#protocol_12 here].
Results:
The graph clearly shows the lysis of the cells after IPTG induction. 0.1mM and 1.0mM are representative for the other measurements taken.
The blue line on the graph corresponds to the uninduced pT7 lysis device which is also representative for the untransformed E. coli control.
Discussion:
The Lysis cassette (BBa_K112808) is improved by adding a pT7 promoter (BBa_I712074) in front of it in the part BBa_K1022114. We showed the lysis device is capable of lysing E. coli efficiently and quick.
The functionality of the lysis device is improved as the promoter provides more controllability to the existing part BBa_K112808.
From the experiment, it can be clearly noted that the cells are growing exponentially and when induced by IPTG, within minutes the lysis device starts to kill the cells. Whereas the control E. coli cells with or without the plasmid used in the experiment, are growing even after IPTG is added to them. This proves that the cells do not die due to the IPTG chemical, but due to the lysis device which is activated by the pT7 promoter.
| n/a | pT7 : Lysis Device |