Team:CSWProteens

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	Team CSWProteens

Team

The Cambridge School of Weston is located in Weston instead of Cambridge, Massachusetts in America. It is a progressive, co-educational, day and boarding school for students in grades 9 to 12 and post-graduate. Since the school's inception in 1886, it has been at the forefront of educational innovation.

Our teachers are passionate. And we are bold, thoughtful, and some of the most interesting people you’ll ever meet. We are The Cambridge School of Weston, CSW Proteens. We will rock the world upside down!

The team consists of 14 intelligent students who stand strong with the best instructors on the earth, Howard G. Goldsweig and Melodie Knowlton. Oh, not gold swag but Goldsweig. It must have meant the same. The team is led by Joey Booties, sorry, Joseph Boots-Ebenfield actually, a courageous young lad who started the iGem team for the school from zero to infinity. The team members, Aiden Kolodziej, Jenny Woo, Matthew Waters, Ellie Wolf, Nate Williams, Joey Boots-Ebenfield, Noa Machover, Ben Klebe, Summer Ardell, Claire Kerper, Thomas Schaffner, Micah Rickles-Young, and Liam Kaufer. As united as the round table knights (if you ignore Lancelot) and as smart as Albert Einstein. Ah! And please don't forget the honored webpage editor Yixuan Liu. The editor was "honored" because he was too lazy to do anything. (Just kidding) :)

Project

The project undertaken by the Cambridge School of Weston (CSW) 2014 iGEM team builds upon the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team which synthesized an “antifreeze” protein (RiAFP) isolated from a cold-tolerant beetle called Rhagium inquisitor. This is the most potent antifreeze protein so far studied. Our device (Plantifreeze) is designed to mitigate crop damage caused by ice crystal formation on the plant surface. Plantifreeze is a dual plasmid system.

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)

To save time and simplify construction, the two parts will be made by direct synthesis and ordered as gBlocks™ from IDT. gBlocks Gene Fragments are custom, double-stranded, sequence-verified fragments of DNA up to 700 bp. gBlocks Gene Fragments are synthesized using the same industry-leading, high-fidelity synthesis chemistries developed by IDT for our Ultramer™ oligonucleotides, and are sequence verified prior to shipping. The high sequence fidelity and rapid delivery time make gBlocks Gene Fragments ideal for a range of synthetic biology applications, including the ability to easily assemble multiple gene fragments to reliably generate even larger gene constructs.

Plasmid #2. The second plasmid, pLG575, carries tet-regulated HlyBD proteins necessary for HlyA-tagged protein secretion. Plasmid pLG575 has p15A origin of replication and carries the chloramphenicol resistance marker.

Cotransformation. The two plasmids are co-transformed into an E coli strain BL21 (DE3) chassis, a bacterial strain carrying the gene for the T7 RNA polymerase. With respect to dual plasmid systems, it is not possible to maintain in a single cell two different plasmids that use the same mechanism for replication. Plasmids with similar origins of replication, and therefore similar systems for regulating the replication and segregation of the plasmid, are said to be in the same “incompatibility group” and cannot both be passed down consistently to daughter cells. Effectively, the two plasmid clones compete for the available factors required for replication and partitioning. Our device uses compatible plasmids in that replication origin for pLG575 is p15A and that for the plasmid that controls RiAFP expression is pMB

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

DEVELOPMENT OF SECRETION SYSTEM

Obtaining a fusion of the protein of interest (RiAFP) with hemolysin C-terminus results in secretion of the protein into the growth medium. A RiAFP expression and secretion system was constructed by cotransforming competent BL21(DE3) cells with a plasmid containing RiAFP:His tag:HlyA and the plasmid pLG575 carrying HlyBD proteins necessary for HlyA secretion.

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%.

DESIGN of gBlocks

Many restriction endonucleases require several nonspeciic nucleotides (nucleotides) on either side of their restriction site to cut, and do not cleave efficiently when the restriction site is located at the immediate end of a DNA fragment. IDT suggests adding 7 bases of sequence between the restriction sites and the free ends of the DNA insert required to allow the restriction enzymes to cut properly.

Overview of the Construction Process for a Dual Plasmid Device

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.

Notebook

Show us how you spent your days.

Results/Conclusions

What did you achieve over the course of your semester?

Safety

Safety is always an important thing for our team. When doing labs, at least one of our instructors is always with us. The lab is well equipped to ensure every team members' safety. We have thick gloves specially designed for preventing things from burning our hands. Goggles to prevent bad things spitting into our eyes. Rubber gloves ensure the sanity and our health. We also have a lab shower and an eye washer that will save our lives if we get into trouble with some dangerous chemicals. Moreover, we all have insurance so we won't pay out of our pockets if something bad happens ;)

Attributions

We've all done a lot of work so far, with more to come! Summer, Micah, and Aiden are our primary fundraisers. They wrote and sent letters to local biotech companies letting them know what we're doing, and that we would love to get some help from them. Lilly has been doing most of the work on our two social media outlets other than the wiki (our tumblr and facebook page). Ellie has been contacting middle schools in the area to see if they would like to have us teach about synthetic biology and DNA. Noa is our project manager, and has been organizing everyone, and keeping the entire team on track. Joey is the team leader, and has been busy coordinating all of the little things there are to do. Everyone has been involved in design and lab work. And of course, Melodie and Howard are our advisors!

Human Practices

Fun!

What was your favorite team snack?? Have a picture of your team mascot?

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