Part:BBa_K2666004

VHH LaM-4 with signal peptid YncM with a L.jensenii specific strong promoter

I. Part BBa K2666000 : Function

The Montpellier iGEM team 2018 designed a construction that produce and secrete LaM-4, a nanobody against RFP and mcherry in Lactobacillus jensenii (more information). For that, we used the promoter RpsU, a L.jensenii specific strong promoter [1]. The signal peptid is B. subtilis specific [2]. Figure 1 illustrates the detailed design.

Figure 1 : Construct design. That sequence encodes VHH LaM-4 with a SP YncM regulated by a strong promoter RpsU.

LaM-4 is used as a control [3]. It is an antibody against RFP & mcherry. This control allow us to check the efficiency of the promoter and the signal peptid.

III. Design Considerations

We added spacers to all of our constructions to unable easier use of the sequence and separation of the different genes of the sequences. We used two Terminators to our sequences :BBa_B0014 & BBa_B0015 to ensure the stopping of the transcription.

Reference:

[1] Bao, Sujin, et al. "Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats." PloS one 8.3 (2013): e60007.

[2] Brockmeier, Ulf, et al. "Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria." Journal of molecular biology 362.3 (2006): 393-402.

[3] Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ... & Rout, M. P. (2014). A robust pipeline for rapid prod

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 62
  Illegal BamHI site found at 194
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]