Part:BBa_K3425018

iGEM Type IIS standard RBS template

This series of iGEM Type IIS templates present easy to use blueprints to design parts for this assembly standard. These parts provide ready-made sequences which contain the different fusion sites for a promoter, RBS, CDS and terminator part according to the following image*.

By adding the fusion and restriction sites by DNA synthesis or PCR, the part is ready to be cloned into the Type IIS universal acceptor pSB1C00. More information about this backbone can be found in its page. It can also be found, together with all our experience in this cloning method, in Team Uppsala 2020's iGEM Type IIS standard guidebook.

*Note: This image was taken from the pSB1C00 page to provide visual aid for the explanation without redirecting to another page.

Usage and biology

This part is an RBS template, and it is used to clone RBS-type parts (such as BBa_B0030) into pSB1C00, the first step towards assembling transcriptional units with iGEM Type IIS assembly. The template is flanked by fusion sites FS_b (TACT) and FS_c (AATG), which will allow it to take the second position when using it for assembly to a Level 1 or pOdd plasmid (such as pSB1K01).

Follow these steps to use this template:

1. Obtain the template sequence from "Sequence and features"
2. Substitute the G homopolymer (annotated as RBS template) by the desired RBS part
3. Add a 5 nucleotide spacer since the assembly scar is five nucleotides shorter than the BioBrick scar. (Refer to BBa_K3425031 for more information)
4. Order the corresponding primers to anneal and extend*.

For ease of use, we provide a .dna file which also contains example primers. This file can be found in Team Uppsala 2020's Parts page.

*Note: These parts are short so they should be ordered as oligos and extended by PCR [1]. The Fw and Rv oligos should overlap by at least 20bp. If the entire part (including fusion sites and SapI sites) is less than 60bp long, each strand can be ordered as an oligo and then annealed directly.

The following table details the part numbers and names of the design templates.

Registry Number	Name
BBa_K3425017	Promoter template
BBa_K3425018	RBS template
BBa_K3425019	CDS template
BBa_K3425020	Terminator template

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 5
  Illegal SapI.rc site found at 39
  

Note: The part contains restriction sites for its cloning into Level 0 universal Type IIS acceptor pSB1C00, therefore it is marked as not compatible with RFC1000 standard, even though it is.

References

[1] https://parts.igem.org/Help:Promoters/Construction#Constructing_a_part_from_synthetic_oligos