DNA Constructs

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DNA Constructs
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DNA Constructs

• Three Types

• Finzi Green
• Kahn A (9C14)
• Kahn C (11C12)

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Finzi Green

• Linear

BUTIts the only DNA that we can clearly see
looping with
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Kahn

• A-tract intrinsic bend in the DNA located near
  the TATA box

• Ideal Operators
• Operators have a very high binding affinity for
  LacI

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Khan A vs C
Kahn A
Kahn C

• A-tract 84 bps

• A-tract 83 bps

• C-C/O-O 156 bps

• C-C/O-O 155 bps

Pretty Similar, eh?
PHASING
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Phasing

• 1 bp 37º turn
• Kahn A has an additional bp in the A-tract and
  different linker regions

Which constructs looping is more ideal?

• Research suggests that Kahn C forms a loop
  slightly easier than Kahn A

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Kahn A vs. CPlausible Loops
THINK 3 basic ways to loop DNA
Requires the most energy to form
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• Finzi Green
• Kahn A
• Kahn C

• Loop Length 305 bps
• Linear DNA

• Loop Length 142 bps
• A track curvature
• 37º more curvature than Kahn C

• Loop Length 141 bps
• A tract curvature

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Expected Results
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Kahn A

• DNA Length 1211 bps
• Loop Length 142 bps
• Effective Shortening 142 bps

• Conversion Factors
• 142 bps 45 nm
• 1pxl 123 nm

We expect 1/3 pxl movement difference between
unlooped (control) and looped DNA
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• Kahn A
• 1/3 pxl change if higher energy loop forms
• Less than 1/3 pxl change if lower energy forms

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Results
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Control
Peak 220 nm
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Protein
Peak 204
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Histogram
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Conclusion
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Conclusion

• What do these results mean?
• The results are hard to interpret because we cant
  see two clear levels of movement

Protein
Control
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Conclusion

• this could mean that there are multiple LacIs
  binding to only one operator.

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THANKS!