Part:BBa_K5034201

pBBR1MCS-terminator plasmid

Description

The pBBR1MCS plasmid is a synthetic plasmid backbone used in various genetic engineering applications in microbial systems. We modified the original pBBR1MCS and added a double terminator rrnBT1-T7Te(BBa_B0015) downstream the Biobrick suffix.

Sequence and Features

This backbone consists of several parts, including promoters, terminators, resistance genes, and prefix and suffix we added.

Figure 1: Plasmid diagram of backbone

Assembly Compatibility:

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

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4981
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2834
  Illegal NotI site found at 4987
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4981
  Illegal BglII site found at 3580
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4981
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4981
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4996
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 562
  Illegal NgoMIV site found at 4244
  Illegal NgoMIV site found at 4527
  Illegal AgeI site found at 402
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Origin

It's antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector.We get the pBBR1MCS-2 plasmid on the addgene, and add a terminator rrnBT1-T7Te(BBa-B0015) to it.

Experimental Characterization and results

We performed double enzyme digestion on the plasmid and obtained a fragment of approximately 5000bp(Fig.2)

Figure 2: Fragment obtained by double enzyme digestion of plasmid backbone

We then introduce it to S.oneidensis.

Chassis and Genetic Context

We successfully expressed in Shewanella onediensis MR-1.

Potential Applications

1. It can be used in microbial fuel cells to study and enhance the extracellular electron transfer capabilities such as in S. oneidensis.

2. It can be used as a backbone for CRISPR-based genome editing systems, enabling precise genetic modifications.

3. It was used in our project to carry components related to phosphorus metabolism. Our results indicate that this component is a good backbone for carrying phosphorus metabolism related components. So it may be able to be used to solve the problems of regulating the electricity production and phosphorus accumulation of S.oneidensis. Thus providing the possibility for organisms to generate electricity while removing pollution.

Reference

1. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995 Dec 1;166(1):175-6. doi: 10.1016/0378-1119(95)00584-1. PMID: 8529885.