Part:BBa_K1205000

Carnitine Dehydrogenase Protein

This part codes for the Carnitine Dehydrogenase protein extracted from Pseudomonas Aeruginosa.

[http://www.metacyc.org/META/NEW-IMAGE?type=NIL&object=CPLX-5221 Carnitine Dehydrogenase], also referred to as CDH, breaks down L-carnitine into 3-Dehydrocarnitine and is part of the unique metabolic system that some bacteria such as pseudomonas aeruginosa apply to use L-Carnitine as a sole carbon and nitrogen source.

Because this biobrick codes for a system of proteins that work by reducing NAD+, we used a spectophotometric assay to test for the reduction activity.

Carnitine Dehydrogenase Reaction

When NAD+ is reduced to NADH, its absorbance at 340 nanometers changes. Using kinetic spectrophotometry we were able to develop curves showing the relative activity of our protein. For these experiments made cytoplasmic extracts for two cell inoculations transformed with our gene of interest and two inoculations with the empty backbone. We then added NAD+ to each cytoplasmic extract, loaded it into the spectrophotometer, then recording the change in absorbance upon adding either HEPES buffer or 1 Molar L-carnitine suspended in HEPES buffer.

L-carnitine NAD+ Assay Results

As you can see from our results, there was a pronounced increase in the biobrick & L-carnitine trial, while the activity in other trials was close to nonexistent.

Quantified Data

Despite the considerable amount of standard error, even when it is taken into consideration there is still a clear increase in NAD+ reduction activity when the protein encoded by our biobrick comes into contact with L-carnitine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 572
  Illegal BamHI site found at 558
  Illegal BamHI site found at 1043
  Illegal XhoI site found at 1518
  Illegal XhoI site found at 1812
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 40
  Illegal NgoMIV site found at 56
  Illegal NgoMIV site found at 121
  Illegal NgoMIV site found at 253
  Illegal NgoMIV site found at 310
  Illegal NgoMIV site found at 451
  Illegal NgoMIV site found at 600
  
• 1000
  COMPATIBLE WITH RFC[1000]