Part:BBa_K1532007
LacI Producer
This part contains a LacI CDS led by a constitutive promoter.
Part Description
Usage and Biology
This device is a LacI provider.
LacI is the repressior of the promoter Plac (BBa_R0011). In most E.coli Strains, the LacI is expressed in a low level, which indicates that there will be a observable leak on the promoter Plac. This leak may be fatal for the whole system if the Plac is used as the starting signor. That's why the over-expression of the LacI is needed in some of the systems.
In this part, we assembled the CDS of the LacI with the Constitutive promoter J23104 and RBS32, which are powerful pump a great amount of LacI for further use.
Function Testing
The RT-PCR was used to test the expression of the LacI.
Materials and Methods
The part was assembled on the PSB1C3 plasmid backbone, and transformed into the competent cell TOP10 (please visit http://2014.igem.org/Team:NUDT_CHINA/Safety for further information about this cell) and cultured in liquid LB media containing 35μg/mL chloramphenicol to OD1.0. The RNA prep was performed with the RNAprep Pure Cell/Bacteria Kit from TianGen(R), the Reverse Transcription was performed with the PrimeScript(TM) RT reagent Kit with gDNA Eraser (Perfect Real Time) from TaKaRa(R).
Primers were synthesized by Invitrogen(R) with the sequence of:
Upper Primer （5'-3'）：GATAGCGGAACGGGAAGG
antisense primer (5'-3'):AGGCGGTTTGCGTATTGG
The PCR was performed with the Recombinant Taq DNA Polymerase TaKaRa Taq(TM) and dNTP mixture (2.5mM each) under the standard 3-step PCR protocol.
The Top10 cell with no plasmid was used as control.
Results
As the result indicates, Bacteria with this part can transcript the RNA of the LacI as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1209
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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