Part:BBa_C0178

autoinducer synthetase for PAI from Pseudomonas aeruginosa (no LVA)

same as C0078 except no LVA tag

New Background Information of LasI by WHU-China 2020

Group: WHU-China 2020

In our project this year, LasI is not only a component in the quorum sensing genetic circuit, but a target protein in the pathogenic bacteria P. aeruginosa for virulence inhibition [1]. Thus we collected information of LasI structure and LasI inhibitors, which is shown in the references [2,3]. Through experiments, salicylic acid, tannic acid and trans-cinnam aldehyde were recognized as potent inhibitors [2].

Figure 1: 3D Structure of LasI [3].

Figure 2: The inhibition of OdDHL production by small molecules [2].

Reference: 1. Grandclement C et al., Quorum quenching: role in nature and applied developments. FEMS Microbiology Reviews, 40 (2016): 86-116.

2. Chang CY et al., Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target. Scientific Reports, 4 (2014).

3. https://www.rcsb.org/structure/1RO5

Characterisation of LasI by Shanghaitech iGEM 2017

Group: Shanghaitech 2017

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]