Part:BBa_K3458005

Apis Mellifera Silk Fibroin 3 With Codon Optimization

This part is an improvement of BBa_K1763000, to let Apis Mellifera Silk Fibroin 3 express inArabidopsis thaliana better.

Usage and Biology

According to the codon preference of Arabidopsis thaliana, our team optimized the codon of BBa_K1763000 to make it more suitable for expression in plants, providing a basis for subsequent research on this part in plants.

The following is an introduction to the biological background of the original part. If you need more information, please pay attention to the team 2015 UCLA’s project.

Silk from Apis Mellifera represents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honey bee silk has certain advantages over spider and silkworm silk (Weisman). The size of the honey bee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non-repetitive, which allows us to synthesize and make modifications to the gene without the complications that are inherent to repetitive DNA sequences. Honey bee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha-helices, and four silk proteins come together to form a coiled-coil structure these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4 (Sutherland 2007). However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers (Sutherland). (quoted from [1])

Characterization

Optimization Report Codon Used Adjustment

Fig. 1 Before Codon Adjustment (CAI: 0.63)

Fig. 2 After Codon Adjustment (CAI: 0.88)

Codon Used Distribution

Fig.3 Before Codon Adjustment

‎Fig.4 After Codon Adjustment

GC Content

Fig.5 Before Codon Adjustment

‎Fig.6 After Codon Adjustment

Western Blot

Fig.7 The result of the western blot of BBa_K3458005

Western Blot can detect target bands of the correct size, which means the optimized Apis Mellifera Silk Fibroin 3 protein can be expressed in Arabidopsis protoplasts.

‎Fig.7 The result of the western blot ofBBa_K3458005

As shown, the amount of ASMF is continuously increasing, and it increases dramatically from the third hour.

Also, we tested the expression level of part BBa_K1763000 before codon optimization in Arabidopsis thaliana (western blot) and submitted the data to the description page of BBa_K1763000. 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
  COMPATIBLE WITH RFC[1000]