IGEM:MIT/2005/InsulinActuator

The Actuator Group
Short Term:
 * choose a flourescent protein (people seem opinionated on this!) and spec out the requirements to make it flouresce to aid the sensor and s.p. group's initial system designs.
 * lay groundwork research for making e.coli spit out a fashionable form of insulin. Also, design tests to show presence of insulin and show world that "we meant to do that."
 * lay groundwork research for making e.coli spit out a fashionable form of anything. ..

Papers
Some insulin Papers. One of our local wisemen, Drew Endy, noted that an apparent world expert on the topic of stuff_export and e.coli is/was Karen Talmadge. She used to hang out at Harvard. Now she runs some serious biotech stuff, but in the 80's she was a postdoc doing some pretty cool research that probably catalyzed her rise in industry. We read (present tense) her papers.


 * An 'internal' signal sequence directs secretion and processing or proinsulin in bacteria. (article not available on web; we will scan and post sometime)


 * Cellular location affects protein stability in Escherichia coli.


 * "Bacteria mature preproinsulin to proinsulin." by Talmadge


 * Eukaryotic signal sequence transports insulin antigen in Escherichia coli.


 * Human insulin production from a novel mini-proinsulin which has high receptor-binding activity


 * Construction of plasmid vectors with unique PstI cloning sites in a signal sequence coding region


 * Insulin Background Information
 * A short description w/insulin pics!
 * More insulin info
 * AA sequence of preproinsulin in humans and other organisms
 * MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN
 * AA sequence of rat preproinsulin I
 * MALWMRFLPLLALLVLWEPKPAQAFVK GHLCGPHLVEALYLVCGERGFFYTPKSRREV</FONT>EDP</FONT>QVP</FONT>QL</FONT>ELGGGPE</FONT>AGD</FONT>LQT</FONT>LALEVAR</FONT>QKRGIVD</FONT>QCCTSICSLYQLENYCN
 * Insulin Receptor info -- positive feedback

"An 'internal' signal sequence directs secretion and processing or proinsulin in bacteria."
maxiney



The Big Picture
Above is a rough picture I drew based on my understanding of the paper. The big picture is that this article is useful because it gives us a sense of how to export proinsulin out of the cell. It does this by attaching a "signal sequence" at the end of the proinsulin gene. More detailed explanation is below.

More in depth
PROINSULIN is essentially a precursor of insulin. Proinsulin contains A and B subunits which, when cleaved and then attached together, will form the INSULIN gene. PREPROINSULIN is proinsulin with a signal sequence attached at the end (A signal sequence is a segment of amino acids at the N terminus of a protein, that enables the protein to be secreted, i.e. to pass through the cell membrane). In the article, the end of this signal sequence is removed, and is replaced by the signal sequence for prepenicillinase, which is an enzyme that is exported out of the cell. The signal sequence of prepenicillinase, therefore, tricks the cell into exporting the proinsulin out of the cell. Before the preproinsulin-prepenicillinase construct is exported, the cell removes the signal sequence, leaving only the proinsulin, which is exported out of the cell

bosworth

I haven't gotten past the abstract, but these kids secreted proinsulin. They "tagged" there proinsulin by fusing a preproinsulin sequence with a penicillinase tag/sequence. Preproinsulin is proinsulin with some amino acids typically used to move the protein out of the roughER (golgiapp.) and is immediately cleaved in prokos. In e.coli, the pre_sequence convinces the membrane to let the protein out. Can't stomach the paper yet though, and would really like to find how the sequence was promoted in the first place. These kid's were not trying to control the rate of insulin.

"Cellular location affects protein stability in Escherichia coli"
"These experiments demonstrate that various insulin antigens are at least 10 times more stable when secreted into bacterial periplasm than when left in the cytoplasm, where they have a 2 min. half life. THUS, the intracellular location of a protein can affect its stability."
 * this paper's findings are less in line with what we are trying to do. The methods they used for excretion are not novel either, it seems. But, it does show that the methods for excretion they used were legit methods.

"Construction of plasmid vectors with unique PstI cloning sites in a signal sequence coding region."

 * constructed a series of plasmids with restriction sites near pre-penicillinase.
 * pre-penicillinase is shown to be a viable signal sequence (for protein secretion).
 * "we have created a series of plasmids derived from pBR322 with unique PstI restriction sites...used these plasmids to study protein secretion in bacteria."
 * this may be a good initial universal exit way.