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 proved the optimized part to have a more stable expression in E.coli through 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 through 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; note that our codon-optimized AmilCP has only 79% DNA sequence identity to the chromosomal codon-optimized AmilCP reported in [1]). In order to conduct a stability assay through growth in liquid culture, the codon optimized basic part and the original native AmilCP part were attached to a constitutive promotor, RBS and a double terminator separately. The two Biobrick parts were ordered as customized genes from IDT inserted in pUCIDT (Amp).

Both vectors were transformed into DH5-alpha E.coli cells through single tube transformation and yielded phenotypically blue colonies for cells containing plasmid with original part and plasmid containing codon optimized part (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 (pUCIDT). 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.

Stability assay through liquid experiment was performed with both inserts in the pUCIDT (Amp) vector. 1 mL of LB with ampicillin (25 ug/ml) was inoculated with one single colony in eppendorf tubes in 10 replicates for each part. In order to allow 10 generations of growth, a 1:1000 dilution was made of the overnight culture and incubated for 24 h. The colour comparison was done through visualization of centrifuged cultures and by analyzing the color intensity we could deduce the stability of the chromoprotein-encoding plasmid.

Figure 3: Results after 10 generations of growth. The upper row represents 10 different culture pellets of the cells with the plasmid containing the codon optimized sequence of AmilCP. The lower row represents 10 different culture pellets with the original native AmilCP (part:BBa_K592009) sequence incorporated in the plasmid.

Already after 10 generations of growth it was clearly visible that the color intensity was better distributed in the cells containing the plasmid with the codon optimized AmilCP sequence. The result confirms that the plasmid containing codon optimized part generates better maintenance of protein expression than the plasmid containing the original native part. The improved stability of expression makes this part a good candidate as a reliable reporter or biosensor. Since this improved chromoprotein Biobrick part is equipped with necessary elements for constitutive expression, Part:BBa_K2669003 is convenient to use for synthetic biologists.

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]

References

[1] Liljeruhm J, Funk SK, Tietscher S, Edlund AD, Jamal S, Wistrand-Yuen P, Dyrhage K, Gynnå A, Ivermark K, Lövgren J, Törnblom V, Virtanen A, Lundin ER, Wistrand-Yuen E, Forster AC. 2018. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, doi 10.1186/s13036-018-0100-0.

[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, Matz MV. 2008. Diversity and evolution of coral fluorescent proteins. PloS One 3: e2680

[2] iGEM11_Uppsala-Swede, Part BBa_K592009