Part:BBa_K4006006

FtnA-FLAG

Background

This part includes a coding sequence for the protein ferritin codon optimized for use in the Chlamydomonas reinhardtii chloroplast as well as a human influenza hemagglutinin (HA) tag for visualization of protein expression in a Western blot. Ferritin is a large protein that takes the form of a hollow sphere, sequestering iron inside of itself. While it is extremely well known as a tool for iron sequestration, it can be used to capture multiple heavy metals. It has also been expressed within the Chlamydomonas reinhardtii chloroplast before with a notable difference in capacity for binding iron.

Cloning into E. coli and Verification

This part was transformed into 5-alpha competent E. coli cells from NEB. The cloning was successful, as the positive control plate showed significantly more colonies than the negative control plate.

Figure 1. The plate on the left is the experimental transformation of tagged ferritin into plasmid backbone MT-pASapI using Golden Gate Assembly. Multiple individual colonies are present on the plate, indicating successful transformation. The plate on the right is the negative control. Little to no colonies are present on the plate, indicating that there should not be high background or incomplete recombination in our experimental plate.

This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. FtnA-Flag was expected to have 2 bands: 5.9 kb and 1.3kb. This is a contrast to the MT-pASapI backbone, which would result in two bands of 4.4kb and 2.3kb. The gel showed three bands for all of the constructs run, the top band being undigested plasmid due to ineffective restriction digest. However, the other bands showed that the inserts contained bands of the appropriate size, and were different from the MT plasmid.

Figure 2. Gel electrophoresis of restriction digest with XbaI and BstXI of the recombinant colonies with tagged ferritin in place of MT. Each of the picked colonies indicate the two expected and distinct bands with exception to the first well, which was noted as a failed cloning.

Transformation into Chlamydomonas reinhardtii

We were unable to successfully transform this construct into C. reinhardtii.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 5
  Illegal SapI.rc site found at 560