Team:WalthamHS BioHawks/Project/Design

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Revision as of 15:02, 19 June 2014

Our stain remover gene, subtilisin, is being synthesized with BioBrick prefix and suffix restriction site sequences. The gene has been delivered in a plasmid with the terminator sequence. It is depicted below.

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

Basic Descriptions:

The promoter and RBS are together in BioBrick Part BBa_J04500, which is the same part used in the practice 3A assembly kit. 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 subtilisin enzyme and is part of the natural subtilisin gene. Subtilisin is a serine protease enzyme that degrade other proteins. BB-suffix is the BiobBick sequence with the appropriate restriction enzyme sites to conform to the 3A Assembly standard. The terminator is BBa_B1006.

Parts 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 digest lactose. However we have replaced those genes with genes that code for subtilisin. LacI codes for repressors that halt transcription because 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 a molecular mimic of lactose and the gene is turned on only when IPTG is present but when the glucose 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 subtilisin, 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 subtilisin genes there is a BB-suffix. 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 (BBa_J04500) 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 subtilisin to be produced then LacI should be absent, glucose should be absent but IPTG should be present.

I'll see of I can find pictures of the synthesized gene construct that was delivered from BioBasic.

OmpA signal peptide is 21 amino acids long. It helps to secrete recombinant enzymes out of the bacteria, where time is an important factor in how much has been secreted. Induction time requires several hours, 4 in the experiment. Normally, it is used to translocate and direct enzymes across the cell membrane of the bacteria; the type of bacteria should not be a major factor, but should also not be ignored. (The experiment used Gram-negative E. coli for over-expression of the test enzymes, using OmpA as the experimental.) For some enzymes, OmpA was equally effective in secretion as the native signal peptides, however this is not guaranteed for all enzymes. Due to the large variety of enzymes that OmpA can direct, it is advantageous to use it for non-native enzyme secretion in transformed bacteria.

I'll find a 3D crystal structure that highlights the active site of the enzyme.

Subtilisin is a protein-degrading enzyme. The peptide bonds are degraded with serine residue at the active site. The enzyme works in a signal-transduction pathway, with Ser-221 cleaving the peptide bond with its partially negative oxygen atom. In commercial use, subtilisin is genetically engineered because detergents and high temperatures easily inactivate the wild type. Subtilisin is used to remove protein based stains. It usually consists of 269 to 275 amino acids and its active from pH 6 to 11, with its major activity from pH 9 to 11. Subtilisin is easily found in the environment and is readily biodegradable. Subtilisin can cause a respiratory allergy in some people when come in contact with it in detergent. Reactions differ with the frequency, magnitude, and duration of the exposure. There is not a lower bench for the allergy currently. Because of the low concentrations in detergent, Subtilisin is not a concern for skin or eye irritation. There are no major health concerns for the use of Subtilisin in laundry and cleaning products. Subtilisin comes from the Bacillus genus. The overall tertiary structure of the species is the same, which means a similar function.