Part:BBa_C0178

autoinducer synthetase for PAI from Pseudomonas aeruginosa (no LVA)

same as C0078 except no LVA tag

Characterisation of LasI by Shanghaitech iGEM 2017

Group: Shanghaitech 2017

In synthetic biology, quorum sensing system (QS system) has been researched as a way of bacteria communication. A whole system always includes two parts: a generator of AHL molecules and a reporter which can receive molecules and activate the downstream genes’ expression. For ‘Las’ system, the LasI coding sequence can be translated into LasI protein, which also works as an enzyme to catalyze the reaction of substrates into Las molecule (3OC12-HSL). According to the previous parts of iGEM before 2017, a little information can be searched, thus this year we construct two devices – ‘Las molecule generator BBa_K2315033 ’ and ‘Rpa-Las molecule converter BBa_K2315046’. The first one can generate Las molecule without induction constitutively, the second one, however, can receive another QS system’s molecule – Rpa. After its induction, Las molecule can be generated, which means that this device can convert Rpa molecule into Las molecule. The mechanism of these two devices are shown below, the circuit design (.dna file) can be download in this part’s ‘design page’, the characterization of them can be found in this part’s ‘experiment page’. In conclusion, we successfully achieve the goal of ‘improve the function of an existing Biobrick Part’ with LasI coding sequence in these ways:

• 1) LasI coding sequence is functional
• 2) With different gene circuit design, LasI coding sequence can be generated by different inputs (constitutive promotor without inducer and pRpa promotor with Rpa molecule as inducer). Furthermore, it can be designed for complex logic circuit – converter is one of the examples.
• 3) QS systems’ AHL molecules (here is Las molecule – 3OC12 HSL) generated by bacteria can be detected by HPLC and LC-MS
• 4) Las molecule has a long half-period time, it is also robust and stable in incubating mixture.
• 5) QS systems’ AHL concentration (here is Las molecule – 3OC12 HSL) can be calibrated by HPLC’s relative peak area.
• 6) LasI’s product can be calibrated by GFP’s fluorescence by plate reader and fluorescence microscope.

In synthetic biology, the quorum sensing system (QS system) has been used intensively for bacteria communication. The QS system has two parts: a generator of AHL molecules and a reporter which receives AHL molecules to activate downstream gene expression. For ‘Las’ QS system, the LasI protein catalyzes the enzymatic production of Las molecule (3OC12-HSL). A part expressing LasI has been deposited for more than a decade (since 2004), yet little information are available. This year this year we constructed two devices to ‘improve’ this part – ‘Las molecule generator BBa_K2315033 ’ and ‘Rpa-Las molecule converter BBa_K2315046’. The first one can generate Las molecules constitutively without any induction; the second one is a ‘converter’ which can receive another QS signal molecule Rpa and then produce Las. The mechanism of these two devices are shown below; the circuit design (.dna file) can be download in the part’s ‘design page’; the characterization of them can be found in this part’s ‘experiment page’. In conclusion, we successfully achieved the goal of ‘improve the function of an existing Biobrick Part’ with LasI coding sequence:

• 1) We verified LasI coding sequence is functional after more than a decade of deposition.
• 2) We used HPLC and LC-MS to quantitatively measure the product of LasI, Las (3OC12 HSL), which has not been done before.
• 3) We verified our two devices can produce Las successfully upon linking LasI to a constitutive promoter or a pRpa promotor induced with Rpa.
• 4) We verified that Las has a very long half-life, very stable in culture mixture.
• 5) We found Las concentration can be reported by real time GFP fluorescence upon mixing with a reporter bacteria when measured quantitatively using a plate reader.

Device 1: The mechanism of Las molecule generator

Device 2: The mechanism of Rpa-Las molecule converter

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 223
  
• 1000
  COMPATIBLE WITH RFC[1000]