Part:BBa_K3904118

p-slpA + sfGFP + TAL

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefor target E. histolytica from several angles: prevention and diagnostics. As a tool to prevent amebiasis, our team created probiotics capable of naringenin biosynthesis. For the diagnostic part, the project includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers, specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

To assure continuous and efficient naringenin production, we had to guarantee the proper expression of naringenin synthesis enzymes in our probiotic strains. To reach the maximum efficiency of the natural synthesis pathway, we decided to manipulate the expression rates of these proteins by finding the promoters of optimal strength for the expression of each of these enzymes using pTRKH2 vector. In addition to that, we also introduced an mRNA cyclization system to this project. The circularization of mRNA molecules is supposed to improve the fraction of full-length proteins among synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation.

Promoter strength was estimated by measuring how intensively the nissle transformants can produce super fold GFP (sfGFP) fused with tyrosine ammonia lyase (TAL) under the promoter of interest. Fluorescence intensity was evaluated by dividing the intensiveness of the signal by the OD600 of the medium during the course of 6 hours (Figure 1). The greater ratio would indicate the stronger promoter (Table 1). The data were compared between all the promoters of our interest and the sequences demonstrating required expression rates were selected for the construction of naringenin synthesis cassete. The same evaluation strategy was applied to measure the effectiveness of mRNA cyclization system, which in our particular case did not show the expected potential to improve the efficiency of our system (Figure 2)[1].

Figure 1: first graph - comparison of the strength of all target promoters; second graph - the strength of all target promoters compared to p-slpA.

Table 1: the evaluation results of the promoters without mRNA cyclization system.

Promoter	Absolute value of fluorescence/OD600 at the midpoint of growth	Promoter strength in comparison to p-slpA
BBa1033225	383.99	0.76
BBa1033222	227.79	0.45
BBa1033220	295.25	0.58
P-slpA	508.22	1.00
J23118	176.43	0.35
J23117	155.74	0.31
J23115	135.46	0.27
J23114	175.06	0.34
J23113	180.56	0.36
J23107	253.16	0.50
J23106	237.59	0.47
J23103	178.62	0.35
J23102	187.82	0.37
J23101	226.14	0.44

Figure 2: comparison of general promoter strength with and without mRNA cyclization system.

Usage and Biology

Lactobacillus acidophilus ATCC 4356 slpA promoter and gene encoding for sfGFP fused with tyrosine ammonia lyase (TAL) gene to evaluate the expression of fused proteins under the aforementioned promoter.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 2458
  
• 12
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  Illegal SpeI site found at 2458
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2267
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 2458
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 2458
  Illegal AgeI site found at 823
  Illegal AgeI site found at 1066
  Illegal AgeI site found at 1423
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 88
  

References

1. Yang, J., Han, Y.H., Im, J. et al. Synthetic protein quality control to enhance full-length translation in bacteria. Nat Chem Biol 17, 421–427 (2021). https://doi.org/10.1038/s41589-021-00736-3