Part:BBa_K3112041

Chromosomal stabilization element group example 2

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

This is our second example of testing chromosome stabilizing elements.The insertion site of this element is about 2K away from the centromere of Saccharomyces cerevisiae.This is also an another attempt to insert new centromere directly into the peripheral heterochromatin region. The aim of this attempt is to ensure that the separation site of this chromosome in the fusion system is consistent with that before fusion without affecting the original cell if possible.

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. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 133
  Illegal PstI site found at 234
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1346
  Illegal PstI site found at 234
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 136
  Illegal BglII site found at 1237
  Illegal BglII site found at 1297
  Illegal BglII site found at 1514
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 133
  Illegal PstI site found at 234
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 133
  Illegal PstI site found at 234
  
• 1000
  COMPATIBLE WITH RFC[1000]

This is an another attempt by us to directly insert new centromeres into the peripheral heterochromatin region. The purpose of this is to ensure that this is in the fusion system without affecting the original cells as much as possible. The location of the chromosomal binding site of the chromosome and the dividing chromosome are consistent with those before fusion.
There is currently no literature on centromere epigenetics that clearly indicates the consequences of doing so, and our follow-up test results may provide some reference for this research.

Figure 1. Insertion position of centromere

Figure 2. Growth status of transformed cells

Figure 3. E. The morphology of transformed cells

Figure 4. 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 5. Phenotypic analysis of three srtins

By comparing the transformants with the original cells, we find that they apparently do not change much in morphology and growth pattern, and the metabolic pathways on this chromosome that can produce lycopene can still be expressed normally.
However, we still find that the growth of the transformants is slower. This may be due to the fact that multiple centromeres on the same chromosome work together during the cleavage phase. Even if they are closely spaced, there may be a phenomenon in which the entire chromosome is broken or cannot be separated from the middle.

Figure 6. CR validation map of transformants (D and E are the two transformants we verified. From left to right are the connection points of L-his, his-cen and cen-R)

Figure 7. Sequencing results of transformant centromere fragments (No. 4 and No. 12)

This shows that the centromere fragment we integrated into S. cerevisiae is correct.