Difference between revisions of "Part:BBa K3149001"

 
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Safety and chemicals
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Aside from microorganisms, we used several chemical reagents, one being ethidium bromide (EtBr). During our experimental procedures, we worked with this reagent to visualize DNA during electrophoresis, and it is believed to be a mutagen and probable carcinogen.
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As an alternative, we employed GelRed, an alternative marker for DNA electrophoresis. GelRed is marked as less toxic and more sensitive than ethidium bromide (EtBr), becoming a priority during these procedures.  
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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.
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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.
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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.
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Representative image of fluorescent colonies corresponding to bacteria transformed with the pOmpC+TRZ1+iLov construct.
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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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 03:43, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 690
    Illegal AgeI site found at 802
    Illegal AgeI site found at 1351
  • 1000
    COMPATIBLE WITH RFC[1000]