Part:BBa_K1716002
NahR Biosensor for detection of acetylsalicylic acid/aspirin with blue chromoprotein reporter
Figure 1 Blue colouring of culture after 24 incubation in LB + 2mM acetylsalicylic acid (right). Culture the left is LB
Design
In order to design a laboratory project that could be done in a high school laboratory with limited access to standard laboratory equipment, the biosensor was created without using gel electrophoresis or gel purification. 3A assembly using another vector backbone could have been used to improve efficiencies of cloning, it is the Danish Ministry of Education interest to limit the number of different antibiotic resistances genes used in the laboratory due to biosecurity. For this reason the pSB1C3 was the only plasmid backbone used in the construction. By cutting the first BioBrick with EcoRI, SpeI, and PstI, a short dsDNA is created that cannot re-ligate back into the vector. Cutting of the second BioBrick with EcoRI, XbaI, and PstI, a similar short dsDNA fragment is generated. If correct successfully, the BioBricks can only assembly into pSB1C3, if they are ligated together. In the process a short scar between SpeI and XbaI is generated (Figure 2). Alternatively, the BioBrick can be inserted using the standard linearised pSB1C3 backbone from the Distribution kit.
Figure 2 From iGEM Registry of Standard Biological Parts. Standard prefix and suffix with restriction sites according to iGEM RFC10.
Methods:
Plasmid purification pSB1C3 BioBrick containing plasmids were isolated from NH5-alpha E. coli cells using QIAprep spin columns. Prior to purification, plasmids were transformed into chemical competent DH5-alpha E. coli and selected on LB agar plates with 6 ug/mL chloramphenicol.
Cloning of Composite BioBrick 500ng of BBa_J61051 and BBa_K592009 was digested indibidually in a total volume of 20uL. BBa_J61051 was digested with EcoRI, SpeI, and PstI, and BBa_K592009 was digested with EcoRI, XbaI, and PstI for 1 hour. After heat activation at 80C for 20 minutes, the two parts where mixed in equal volumes with T4 DNA ligase and incubated for 15 minutes at room temperature. 1uL of ligation reaction was used to transform DH5-alpha chemical competent cells. Transformation reaction were plated on 6 ug/mL chloramphenicol containing LB agar plates.
Evaluating biosensor Transformants were selected and overnight cultures containing 6 ug/mL chloramphenicol were started. The following morning, the cultures were diluted to OD600=0,2. Different concentrations of acetylsalicylic acid was added by adding the drug kodimagnyl in different concentrations to LB media.
Results
The biosensor was cloned as described above. 8 transformants were selected and grown overnight. The next morning, the cultures were diluted to OD600=0,2. 0, 200uM, and 2mM acetylsalicylic acid was added to the innoculated culture. After 24 hours the colour of the culture was analysed. Half of the cultures had a dark/blue color which was only detectable by eye when the concentration of acetylsalicylic acid was 2mM (Figure 1).
Additional information
The NahR transcription factor is able to detect a range of other compounds. To read more about this, please refer to [http://2013.igem.org/Team:Peking/Project/BioSensors/NahR Peking iGEM 2013 Wiki]. The biosensor was also tested with GFP and YFP as reporters instead of amilCP. Both reporters could be detected using fluorescence spectroscopy (data not shown).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 786
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 77
Illegal NgoMIV site found at 618 - 1000COMPATIBLE WITH RFC[1000]
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