Part:BBa_K639000
LacI-pLac-mCherry with RBS and terminator
This brick will yield red colonies as it is. When a promoter is inserted in front of the brick, the red color will disappear or be weaker, due to production of LacI that repress lac-promoter. One way of using this brick is to insert a promoter that you suspect, or know is repressed by a molecule or other stimulus. When the promoter is inhibited, transcription of LacI will stop and mCherry will be produced. Thus, the stimuli can be directly measured as intensity of red colour or fluorescense.
Because this construct lacks a downregulated promotor in front of it, LacI will not be syntethized, and in response, pLac will not be downregulated of LacI. Since pLac is not downregulated, mCherry is going to be present whether the cells are stressed or not.
Our experiments indicate that the biobrick is put together the right way.
The stress sensor precursor is made from the following biobricks:
| Biobrick | Part number |
|---|---|
| LacI with RBS | BBa_J24679 |
| Terminator | BBa_B0015 |
| pLac | BBa_R0011 |
| mCherry with RBS and terminator | BBa_J06702 |
The composite biobrick has a total length of 2153 bp, and was originally located in the plasmid pSB1A2.
In order the check if the construct was correct we digested the plasmid with BsaAI + HpaI and BstBI SpeI.
Prior to shipping the biobrick was transfered to the official shipping plasmid pSB1C3.
This construct could in theory be used for any kind of biosensor. The only thing needed is a promotor that gets downregulated directly or indirectly by the conditions one would want to detect. One example is the stress sensor we made BBa_K639003
Sequencing
Sequencing confirmed the sequence of the BioBrick.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1165
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |