Part:BBa_K3112042

Chromosomal stabilization element group example 1

Our chromosome stabilizing elements consist of two parts: centromeres and tag for screening.

In this example, we try to directly replace the centromere of Saccharomyces cerevisiae.

Before chromosome transferring, we hoped the element group will not have a great impact on the growth and division of cells. It can stabilize this chromosome with stable elements when the other organism of the fusion system is dominant.

In order to meet the relevant iGEM standards for part, we deleted the homologous arms of Saccharomyces cerevisiae on both sides of the part. That doesn't mean we did the same in the experiment. The significance of creating this example is that we hope that later teams can add the stable element of chromosome that can affect cell growth to any position through PCR or other means.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 906
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 797
  Illegal BglII site found at 857
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

After obtaining the results of the above two experiments and conducting relevant analysis, we have new ideas. Comparing the Saccharomyces cerevisiae with the centromeres of the Yarrowia lipolytica, we can see that there is not much difference between them overall. They both have point centromere, and the two yeasts are closely related, and there is no clear evidence that Saccharomyces cerevisiae cannot recognize the centromere of the Yarrowia lipolytica. So after replacement, we will be able to obtain a Saccharomyces cerevisiae that uses the Yarrowia lipolytica centromere to direct chromosome-dissociation-related behavior.

Figure 1. Location of the centromere replaced (the position between the left and right homology arms)

Figure 2. The morphology of transformed cells (on the left is Saccharomyces cerevisiae, on the right is Saccharomyces cerevisiae inserted the centromere of Yarrowia lipolytica)

Figure 3. Growth curve of transformants versus original cellsa
(a.Saccharomyces cerevisiae b.Saccharomyces cerevisiae inserted into the Yarrowia lipolytica centromere c.Saccharomyces cerevisiae inserted the centromere of Yarrowia lipolytica)

Figure 4. Phenotypic analysis of three srtins

From the growth status of Saccharomyces cerevisiae, the morphology and growth of the transformants and the manner of division did not change much, and the metabolic pathway capable of producing lycopene on this chromosome could still be expressed normally. It shows that the whole cell can still grow normally after replacing the centromere.

Figure 5. PCR validation map of transformants

Figure 6. Sequencing results of transformant-related fragments

This shows that the centromere fragment we integrated into Saccharomyces cerevisiae is correct.
We also compared the lycopene production (by comparing the color) of yeast inserted with centromere, yeast replaced with centromere and original Saccharomyces cerevisiae. It was apparent by visual observation that the yield of Saccharomyces cerevisiae with an additional heterologous centromere was significantly lower than the other two, and the Saccharomyces cerevisiae replacing the centromere was similar to the original yeast.