Part:BBa_K2669003

Strong constitutive promotor + RBS + Optimized Amil CP (BBa_K2669002) + double terminator

Blue chromoprotein Biobrick part containing codon optimized original native AmilCP Part:BBa_K2669002, RBS Part:BBa_J34801, constitutive promotor Part:BBa_J23119 and a double terminator Part:BBa_B0014.

Usage and Biology

AmilCP is a blue chromoprotein that originates from the coral Acropora millepora, which naturally exhibits strong color when expressed that can be observed with naked eye in both LB and agar culture. The chromoprotein can be used as a quantitative reporter. By performing codon optimization on original native AmilCP, the team of Uppsala 2018 have improved the translation efficiency and prooved the optimized part to have a more stable expression in E.coli trough several generations of growth.

The chromoprotein was first codon optimized by Austin Texas 2017 iGEM team. By performing codon optimization on the seemingly unstable original native AmilCP from 2011, they hypothesized that the chromoprotein would get increased translation efficiency in E.coli. The Uppsala 2018 team have conducted another codon optimization on the original native AmilCP in order to get an expression that can be maintained for a longer period of time trough several generations of growth, hence making the expression of the blue chromoprotein more stable.

Eperimental Design

The original native AmilCP sequence, Part:BBa_K592009 was codon optimized for E.coli K12 with a codon optimization tool provided by Integrated DNA Technologies (IDT).

In order to conduct a stability assay through growth in liquid culture, the codon optimized basic part and the original native AmplCP part was attached to a constitutive promotor, RBS and a double terminator separately. The two Biobrick parts was ordered as gBlocks from IDT inserted in a cloning vector with ampicillin resistance.

Both vectors was transformed into DH5-aplha E.coli cells through single tube transformation and yielded phenotypically blue colonies for both cells containing plasmid with original part and plasmid containing codon optimized part (see Figure 1 and 2).

Figure 1: Transformation plate of colonies with the incorporated plasmid containing the IDT optimized original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the stability assay are circled.

Figure 2: Transformation plate of colonies with the incorporated plasmid containing the original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the assay are circled.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]