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Team:CSWProteens/project
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| + | <b> Promoter. </b>Our device uses a T7 system, as did the Yale team, because T7 RNA polymerase is an incredibly fast and powerful enzyme transcribing rapidly and profusely for as long as the T7 RNA polymerase is present. It synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently. Expression can only be achieved in a bacterial strain carrying the gene for the T7 RNA polymerase. The most common cell strain to use with a T7 promoter system is BL21(DE3) (competent cells are chemically competent cells used for high-level protein expression with T7 RNA polymerase-based expression systems). The BL21(DE3) contains the T7 RNA polymerase gene, under the control of the lacUV5 promoter, integrated into the chromosome. IPTG is used to induce the expression of recombinant proteins cloned into vectors downstream of a T7 RNA promoter and transformed into the BL21(DE3) cells. | ||
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| + | The T7 promoter is a BioBrick part BBa_1712074<p> | ||
| - | < | + | <b>C-terminal His-tag.</b> In our protein expression construct, there is an amino acid motif that consists of six histidine (His) residues fused to the C-terminus of RiAFP. Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. The polyhistidine-tag is to be used to detect the secreted protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. |
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| + | <b>HlyA-signal peptide. </b> The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target RiAFP for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to RiAFP results in transport of the protein from the cytoplasm to the extracellular medium in a single step.<p> | ||
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| + | The HlyA signal peptide is BioBrick pat BBa_K208006<p> | ||
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| + | <b>Terminator.</b> There are several E. coli transcriptional terminators available via the Registry. The most commonly used type of terminator is a forward terminator. When placed downstream of a genetic part that is transcribed, a forward transcriptional terminator will cause transcription to abort. We use part BBa_B0015, a double terminator (B0010-B0012). BBa_B0015. This terminator has an Average Forward Termination Efficiency of 98.4%. | ||
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Revision as of 02:18, 13 June 2014
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Abstract:
Annually, about 5% to 15% of agricultural produce is lost due to frost. The formation of ice damages plants by rupturing cells and also through dehydration as water molecules are drawn out of tissue. Current solutions to this problem - such as using heat or covering crops with protective material - are cumbersome, costly, and not fully preventative. Synthetic antifreeze chemicals have not been proven to work. Even if they did, they would be need to be applied repeatedly, at great cost, and may also leave residues in the environment. The CSW ProTeens aim to design a synthetic biology solution to this problem with a nonpathogenic strain of E. coli able to produce and secrete RiAFP (Rhagium inquisitor Antifreeze Protein) using a part that the Yale ‘11 iGEM team developed. RiAFP is an insect antifreeze protein from Rhagium inquisitor and the most efficient AFP known. RiAFP, unlike synthetic chemicals, should not be harmful if ingested. We are not sure yet whether this method is effective on an industrial scale and will need to test this. We wish to use a type 1 secretion system designed by the Utah State University ‘09 iGEM team to transport the protein directly to the extracellular space. Plantifreeze is a dual plasmid system. Our device is designed to mitigate crop damage caused by ice crystal formation on the plant surface. |
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Plasmid #1. One plasmid controls RiAFP expression which is tagged with a 6x His motif and a HlyA secretion signal. RiAFP/6xHis/HlyA is a fusion protein. The second plasmid, pLG575, carries HlyBD proteins necessary for HlyA-tagged protein secretion. Initiation of transcription of RiAFP/6xHis/HlyA is controlled by a phage promoter, T7. A double terminator stops transcription. Thus, the genetic circuit contained in plasmid #1 is assembled from two parts and must adhere to the biofusion standard: • Part A : 7T-RiAFP/His (no stop codon) • Part B : HlyA/double terminator (no start codon) |
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Promoter. Our device uses a T7 system, as did the Yale team, because T7 RNA polymerase is an incredibly fast and powerful enzyme transcribing rapidly and profusely for as long as the T7 RNA polymerase is present. It synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently. Expression can only be achieved in a bacterial strain carrying the gene for the T7 RNA polymerase. The most common cell strain to use with a T7 promoter system is BL21(DE3) (competent cells are chemically competent cells used for high-level protein expression with T7 RNA polymerase-based expression systems). The BL21(DE3) contains the T7 RNA polymerase gene, under the control of the lacUV5 promoter, integrated into the chromosome. IPTG is used to induce the expression of recombinant proteins cloned into vectors downstream of a T7 RNA promoter and transformed into the BL21(DE3) cells.
The T7 promoter is a BioBrick part BBa_1712074 C-terminal His-tag. In our protein expression construct, there is an amino acid motif that consists of six histidine (His) residues fused to the C-terminus of RiAFP. Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. The polyhistidine-tag is to be used to detect the secreted protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. |
HlyA-signal peptide. The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target RiAFP for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to RiAFP results in transport of the protein from the cytoplasm to the extracellular medium in a single step. The HlyA signal peptide is BioBrick pat BBa_K208006 Terminator. There are several E. coli transcriptional terminators available via the Registry. The most commonly used type of terminator is a forward terminator. When placed downstream of a genetic part that is transcribed, a forward transcriptional terminator will cause transcription to abort. We use part BBa_B0015, a double terminator (B0010-B0012). BBa_B0015. This terminator has an Average Forward Termination Efficiency of 98.4%. |
