Part:BBa_K2273107

Evaluation Vector with P_xylA to screen for protein specific secretion efficiency

Figure 1: Vector map of the EV. The Multiple Cloning Site is indicated in colors, grey elements refer to features necessary for cloning in E. coli and the white elements refer to B. subtilis specific vector parts..

The Evaluation Vector (EV) is part of both, the [http://2017.igem.org/Team:TU_Dresden/Composite_Part Evaluation Vector] and the [http://2017.igem.org/Team:TU_Dresden/Composite_Part Signal Peptide Toolbox] of the [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)].

The EV was developed with easily exchangeable units: I) allowing the replacement of the promoter (which drives the system) and II) a multiple cloning site enabling to work with translationally fused composite parts.

In summary, the EV was designed to fulfill the following distinct features:

1. Exchangeable promoter region
2. Insertion of basic or composite parts as expression units
3. Fulfilling the RFC10 and RFC25 BioBrick standard
4. Easy cloning and screening procedure in Escherichia coli

We constructed the Evaluation Vector (EV) to quickly screen for the secretion of a protein of interest. This vector contains a specifically designed multiple cloning site (MCS) equipped with reporters to quickly identify positive replacements by insert integration (Figure 1). Additionally, this vector can be applied for the expression of any other fusion protein of interest regulated by a promoter of your choice. For more details on the application check out our project.

In the case of the Signal Peptide Toolbox which was designed to increase protein secretion efficiency, a typical composite part consists of a signal peptide for secretion in bacteria (for example AmyE) and a protein of interest.

Sequence and Features

The DNA sequence of this part has been verified via sequencing before it was sent in.

Assembly Compatibility:

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

  Illegal BamHI site found at 247
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1354
  Illegal AgeI site found at 1348
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 266