Part:BBa_K3904504

Seq In 1.4

Aptamers that have been evolutionized during SELEX are mainly sequences by Sanger or NGS. The last-mentioned method applies for High-Throughput SELEX and in this part aptamers need to undergo addition of adapters, primers and other specific sequences. Range BBa K3904501 - BBa K3904520 is particularly for indexing of different SELEX rounds.

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part 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.

Next Generation Sequencing

This part applies for sequencing in MiSeq. To use indexing in another platform check for the corresponding protocol.

Indexing

In table 1 provided indexes can be combined to pool 100 different samples. For Vilnius-Lithuania iGEM 2021 team indexes were used only once.

Table 1. Indexes for different SELEX rounds

Indexed sequencing
No	5' end	3' end
1	Seq_In_1.1	Seq_In_2.1
2	Seq_In_1.2	Seq_In_2.2
3	Seq_In_1.3	Seq_In_2.3
4	Seq_In_1.4	Seq_In_2.4
5	Seq_In_1.5	Seq_In_2.5
6	Seq_In_1.6	Seq_In_2.6
7	Seq_In_1.7	Seq_In_2.7
8	Seq_In_1.8	Seq_In_2.8
9	Seq_In_1.9	Seq_In_2.9
10	Seq_In_1.10	Seq_In_2.10

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 20
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Illumina. (2020). Indexed Sequencing Overview Guide.
2. Illumina. (2018). Index Adapters Pooling Guide.
3. Illumina. (2019). Illumina Adapter Sequences.