Team:CSWProteens/project

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.

• Part A : 7T-RiAFP/His (no stop codon)

• Part B : HlyA/double terminator (no start codon)

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.

Biofusion standard.

Because RiAFP : His : HlyA is a fusion protein consisting of three domains, we need to do in-frame assembly. BioBrick™ standard assembly is not well-designed for this task because the scar sequence formed by the SpeI-XbaI ligation is 8 base pairs long, so assembly of protein domains causes frameshifts. Pam Silver's lab has developed Assembly standard 23 (RFC 23), often called the Silver standard, for assembling protein domains. It relies on shortening the BioBrick prefix and suffix each by 1 base pair such that the resulting SpeI-XbaI scar is only 6 base pairs long and protein domains can be assembled in frame. In accordance with BBF RFC 23, domains of fusion proteins are formatted to contain the prefix 5-GAATTCGCGGCCGCTTCTAGA-3 and the suffix 5-ACTAGTAGCGGCCGCTGCAG-3. Other rules for assembly of fusion proteins include:

• do not start with ATG (Although it is advised to remove the start codon from BioFusion parts to prevent errant translation and formation of unfused proteins, the ATG of the RiAFP sequence was retained so that this coding region could be directly used for C-terminal fusion with the His tag and HlyA signal peptide)

• do not end with a stop codon

• do not have a A or G nucleotide between the end of the XbaI and the beginning of the protein coding region or after the coding sequence and the start of the SpeI

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%.

1) Resuspension of gBlocks™ Gene Fragments (part A and part B}
2) Preparation of pLG575 (depends on format in which the plasmid is received)
3) Prepare cohesive ends of part A, part B, and pSB1A3 construction vector (restriction digestion)
4) Fuse parts A and B into fusion protein and ligate into pSB1A3 (ligation)
5) Transform competent cells with ligated construction vector (transformation) and with pLG575. The selection markers for the two plasmids must be different.
a. pSB1A3 (ampicillin resistance marker)
6) Grow up transformed cells so there are ample copies of :
a. pSB1A3 construction vector
b. pLG575 tet-regulated HlyBD proteins
7) Harvest plasmid DNA from transformed cells carrying pSB1A3 and from cells carrying pLG575 (miniprep)
8) Co-transformation of pLG575 and pSB1A3 into competent BL21 (DE3) E coli. The competent cells will be transformed with one plasmid at a time. Remember each plasmid must have a different selection marker. For transformation of the second plasmid, the plate must contain the selection markers for both plasmids
9) Grow up the dual transformed BL21 (DE3) cells so we have an ample supply of the complete PlantiFreeze device.