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From 2014hs.igem.org

Our stain remover enzyme, subtilisin, is being synthesized with biobrickprefix and suffix restriction site sequences. The gene has been delivered in a plasmid with the terminator sequence also (both with the restrictions site prefix and suffix as found at http://parts.igem.org/Help:Prefix-Suffix) and is depicted below by everything within the parenthesis.

(Promoter + RBS) + (BB-prefix + OmpA + Pro-sequence +Subtilisin + Terminator + BB-Suffix)

•The promoter and RBS are together in biobrick part BBa_J04500, which is the same part used in the practice 3a assembly. •BB-prefix is the Biobrick sequence with the appropriate restriction enzyme sites to conform to the 3A assembly standard. •OmpA is a short signal peptide that coaxes e. coli to secrete the downstream protein out of the cell. •Pro-sequence is a protein that helps guide the folding of the subtilisinenzyme and is part of the natural subtilisin gene •Subtilisin is a serine protease enzyme that degrade other proteins •BB-suffix is the Biobrick sequence with the appropriate restriction enzyme sites to conform to the 3A assembly standard. •The terminator is BBa_B1006

Each Part Explained:

The Promoter is taken from the Lac Operon which is an inducible operon.A Lac Operon is a series of genes that code for proteins that diegest lactose. However we have replaced those genes with genes that code for subtilisin. LacI codes for repressors that halt transripion becasue they prevent RNA polymerase from binding. It can be regulated because it is CAP sensitive. It has two binding sites, the first one allows the CAP protein to bind, while the second one binds to the LacI protein. The IPTG is the molocular mimic of lactose and the gene is turned on only when IPTG is present but the gluocose is absent, otherwise the gene cannot be turned on. RBS is the ribosome binding site, which is where the ribosomes bind together to begin translation. The goal is to translate a protein called Subtilism, which is a serine protein enzyme that is responsible for digesting protein molecules. Before the operon, there is a BB prefix and after the termination sequence to cease transcription of Subtilism genes, there is a BB sufix. The two BB sequences are restriction sites where the the gene can be cut to be incorporated into a plasmid. The Promoter and RBS together is called Part A and that is 220 base pairs. So since the operon we are using is inducible, so when there is no glucose, there is an accumulation of cAMP which binds to CAP and which in turn allows transcription to occur. Also when when LacI is absent, the repressors are not produced and so transcription is possible. So if we want subtilism to be produced there LacI should be absent, Glucose should be absent but IPTG should be present.