Difference between revisions of "Part:BBa K3149001"
Rafael elu (Talk | contribs) |
Rafael elu (Talk | contribs) |
||
| Line 8: | Line 8: | ||
We designed a new part (pompC+Trz1) to evaluate the impact in cell viability of the overstimulation of the endogenous EnvZ/OmpR system, through the establishment of a positive feedback loop between the Trz1 receptor and the ompC promoter. This construct was designed as an improvement of the existing TRZ1 receptor, which is already submitted to the iGEM registry. The co-expression of TRZ1 with the fluorescent reporter iLov allows rapid screening of transformants directly on the plate as shown in the image below. | We designed a new part (pompC+Trz1) to evaluate the impact in cell viability of the overstimulation of the endogenous EnvZ/OmpR system, through the establishment of a positive feedback loop between the Trz1 receptor and the ompC promoter. This construct was designed as an improvement of the existing TRZ1 receptor, which is already submitted to the iGEM registry. The co-expression of TRZ1 with the fluorescent reporter iLov allows rapid screening of transformants directly on the plate as shown in the image below. | ||
</p> | </p> | ||
| + | https://2019.igem.org/wiki/images/d/d1/T--TecMonterrey_GDL--Results-8.png | ||
<p> | <p> | ||
Products from Digestion & Ligation by NEB protocols. 10kb: Quick-Load Purple 2-log DNA Ladder (10kb), L1: pSB1C3 + pBAD-TRZ1, L2: pSB1C3 + pOmpC-TRZ1-iLOV. | Products from Digestion & Ligation by NEB protocols. 10kb: Quick-Load Purple 2-log DNA Ladder (10kb), L1: pSB1C3 + pBAD-TRZ1, L2: pSB1C3 + pOmpC-TRZ1-iLOV. | ||
</p> | </p> | ||
| + | https://2019.igem.org/wiki/images/4/48/T--TecMonterrey_GDL--9-results.png | ||
<p> | <p> | ||
10kb: Quick-Load Purple 2-log DNA Ladder (10kb), M1: Miniprep NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV], D1: NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV] digested with ECORI-HF. | 10kb: Quick-Load Purple 2-log DNA Ladder (10kb), M1: Miniprep NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV], D1: NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV] digested with ECORI-HF. | ||
</p> | </p> | ||
| + | https://2019.igem.org/wiki/images/4/47/T--TecMonterrey_GDL--iLOV.png | ||
<p> | <p> | ||
Representative image of fluorescent colonies corresponding to bacteria transformed with the pOmpC+TRZ1+iLov construct. | Representative image of fluorescent colonies corresponding to bacteria transformed with the pOmpC+TRZ1+iLov construct. | ||
</p> | </p> | ||
| + | https://2019.igem.org/wiki/images/4/40/T--TecMonterrey_GDL--Results-gel-vertical.png | ||
<p> | <p> | ||
Revision as of 03:44, 22 October 2019
TRZ1 receptor under the control of the pOmpC promoter with iLov fluoresent reporter
We designed a new part (pompC+Trz1) to evaluate the impact in cell viability of the overstimulation of the endogenous EnvZ/OmpR system, through the establishment of a positive feedback loop between the Trz1 receptor and the ompC promoter. This construct was designed as an improvement of the existing TRZ1 receptor, which is already submitted to the iGEM registry. The co-expression of TRZ1 with the fluorescent reporter iLov allows rapid screening of transformants directly on the plate as shown in the image below.
Products from Digestion & Ligation by NEB protocols. 10kb: Quick-Load Purple 2-log DNA Ladder (10kb), L1: pSB1C3 + pBAD-TRZ1, L2: pSB1C3 + pOmpC-TRZ1-iLOV.
10kb: Quick-Load Purple 2-log DNA Ladder (10kb), M1: Miniprep NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV], D1: NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV] digested with ECORI-HF.
Representative image of fluorescent colonies corresponding to bacteria transformed with the pOmpC+TRZ1+iLov construct.
In this SDS-PAGE a total protein extraction run is shown to assess the expression of TRZ1 in normal (5g/L NaCl) and high osmolarity (10g/L NaCl) at different dilutions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 690
Illegal AgeI site found at 802
Illegal AgeI site found at 1351 - 1000COMPATIBLE WITH RFC[1000]