Team:UC Davis/Adding secretion

General model for secretion system:

            The purpose of the secretion system is to introduce a method of secreting target proteins we wish to synthesize in the marvelous host: E.coli. Park et. al showed that the truncated form of ice nucleation protein (INPNC) containing the complete coding region of phaZ1, including its signal sequence, could lead to stable secretion of the enzyme (15). We have decided to see if we could co-opt this system to make a more generalized inducible system for protein secretion in which the INPNC and phaZ1 signal sequence serve as carriers for a target protein.

Possible challenges:

1. Efficient recognition of the cleavage site may require some of the non-signal sequence portion of the phaZ1 protein (we have only cloned the signal sequence up to the cleavage site).
2. The system may only work for proteins in a narrow site range.
3. The expression levels of the system may be low.

Advantages:

1.   In our secretion system, we are using two genes with different sizes as our target secretion genes; GFP being short in length and Luciferase being comparably long, these would test the ability of our secretion system to secrete proteins of different sizes.
2.   Testing various combinations of the Signal Sequence with either ompA or INPNC would help us find the best combination for our secretion system based on their secretion ability and allow us to rank the combinations from strongest to weakest in strength. (in this specific system).
3.   We have submitted the BioBricks to the parts registry; therefore they can be used in future studies (for different purposes).

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Secretion Models:
Click on a specific model for more information:

   

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Why test different genes in our secretion system?

     There is wide range of genes present in our environment. Therefore, it is important to measure our secretion system’s ability to secrete genes of various sizes.

Molecular data on some proteins

Name	Molecular Weight (kD)
Myosin	200.0
-Galactosidase	116.3
Phosphorylase b	97.4
Ovalbumin	45.0
Carbonic anhydrase	31.0
Aprotinin	6.5
Insulin, B chain, oxidized	3.5