20.109(F07): Genome engineering assessment

Portfolio components

General notes:

1. email these as .doc
2. email them to nkuldell, endy, nlerner and astachow AT MIT DOT EDU
3. use the following format to name your files: yourfirstname_yourlastname_Mod1_Pt1.doc, so you'll send four files: Bill_Gates_Mod1_Pt1.doc, Bill_Gates_Mod1_Pt2.doc, etc....

Part 1: Rebuttal to editorial

This will be written as a homework assignment, exchanged with your lab partner for peer review, then submitted to the teaching faculty as part of your portfolio

Part 2: M13.1 redesign description and parts

Redesign Ideas from 20.109(S07)

Gene	Ideas
II	make p2 sensitive to and perhaps degraded by any one of various stimuli (e.g. heat, light, pH, chemical input) so replication can be regulated. encode so that p2 can be switched on and off based on the environment which it's in, for example: stops replication of phage genome when in a certain concentration of Ca2+ enters the cell make p2 require a cofactor that must be added before replication of the phage begins make p2 count the number of times it nicks DNA modify p2 in a way that helps p5 sequester the + strands more effectively, perhaps making p5 work more efficiently by reducing competition. modify such that it not only nicks the double stranded form of the genome to initiate replication of the + strand, but also nicks the - strand to impede the formation of dsDNA (this would also help p5). separate g2 and g10 by inserting entire sequence of g10 after transcription end of g2/g10 (essentially repeating g10 twice in succession), so that the two genes are independent alter the gene to make it more active and thus replicate DNA more frequently- see how increased DNA production affects phage growth modify so it can nick foreign DNA (e.g. the E. coli's) and the phage can replicate and package a portion of the host DNA remove p2 and p5 to see if the bacteria still make the phage gene products without replicating the phage DNA
X	make p10 sensitive to a different stimulus than p2 to again regulate replication. perhaps a dual control mechanism for p2 and p10 expression increase the number of p10 so that the phage can produce more double strands. modify such that the + strands of DNA are not soley dependent on the presence of p10. This modification works together with our modification of p2. put a tag on p10 to see what it binds at various parts of replication. This will help elucidate how it controls the amount of double stranded M13 genomes. modify to add another level of regulation for phage propagation. This, coupled with control of II, could allow complex control of the life cycle behavior of the virus. make pX more active so that more + strands will accumulate, allowing the host cell to produce even more phages. extract from gene II.
V	add a tag different from p8 (e.g. RFP) to determine what stage of the phage life cycle it is in or to monitor levels of p5-ssDNA complex. alter interaction with p9/p7 so that a limited number of ssDNA may be surrounded by p8 at a time. make it sensitive to mechanism that allows for assay of DNA amount and location, change its assembly mechanism to influence phage size modify protein so that similar proteins, other than p8 can bind to the surface. remove overlap of p5 gene with start codon of gene for p7. An activation site could be added in prior to start codon in gene for p7 that could be used to allow only limited amounts of pV ( and pVII) to be expressed. This could give control over the the quantity of fully assembled phage to be produced by the host. modify such that p5 can sequester the + stranded DNA more effectively so that there is less competition with the formation of double stranded vary the activity of the protein and thus the competition between dsDNA formation and the sequestering of ssDNA- compare the results to find the optimum level of phage production possible modify expression so that we can control the amount of time the virus DNA spends inside the host (as opposed to actively being packaged and spreading to other bacteria). allow it to sequester double stranded DNA also, then it can be used as a vector for infecting cells with desired DNA fragments. add more DNA binding sites to see if more DNA can be packaged into phage. add some base pairs between V and VII to allow for a restriction site.
VII	alter gene so protein adopts different/more flexible conformation. A change in conformation might expand the different residues that can be attatched to its N-terminal portion. change the way p7 interacts with p9 modify to increase rate host will shed phage, i.e. decrease the phage-host interaction time. modify the last few codons to remove overlap with the gene encoding p9. add sequence to the 5' end of gene so protein could build nanowires or long filaments or other useful materials. delete to learn more about its function. increase it's expression to learn more about its function. tag protein to monitor interaction with p5/DNA complex.
IX	modify p9 to bind to p3 to create long filaments of phage lined up end to end. modify to express different reactive chains on the phage surface. change the function so that p9 will now lyse the bacteria. modify to make the phage secretion occur at a faster rate so that interaction time with the host is reduced. modify beginning and end sequences so that g9 does not overlap with g7 and g8. modify the p9 so that it can bind to bacterial surface proteins (the way p3 does)- see if this allows the phage to interact with other bacteria (now that both ends of the phage can bind and perhaps bridge the two bacterial cells) what is the role of the blunt end of the phage? Why not just have P3 on each side of the filament? We could try to test this in order to try to make the filament more versatile by making matching ends would like to make a phage invading two pili at once. This could create a crosslinker between two cells. It would not be very rigid, but could be bolstered by covering with some materials. The problem is that phage wants to exit with p9 and p7. So a simple idea to create a single particle would be to create two populations of phages which has sticky heads. Upon mixing them we could check if the phages can join to two pili and bring two cells togather. However, it cannot be done with shaking. Modify resulting in faster secretion of DNA
VIII	Add myc, or other tags, e.g. GFP. phage coat protein (2700 copies): alter genes so that the protein P8 has an affinity for certain residues or salts. This can vastly increase the function of m13 as a whole. It can be used to transport different things into bacteria. present small molecules (such as myc epitope), regulate size of phage or influence shape of phage by changing how it assembles into a coat, this could involve changing its interactions with V Make fewer copies so that the proteins can be more flexible. pV and pVII's stop and start codons overlap. Codons could be inserted in between these genes to be able to isolate production of these proteins and controls could be added to their expression levels via activation or repression sites. pVIII expression could be used as a control of maximum genetic length of the phage genome, as pVIII must cover the genome before it is excreted. Add myc or alternative tag to aid in targeting various types of hosts. Add myc or alternative tag to possibly aid in targeting various qualities of host add myc or another tag (x-ray sensitive, UV sensitive, flourescent) Change amino acid sequence to allow g8 (and thus the entire virus) to bind to certain materials, like metals Insert a sequence that shifts g8 so there is no overlap with g9. Then, experiment with various tags to see how receptive the coat is to tagging. Also, could find out whether M13 has a particular predilection for a marker that would allow it to be used for building nanomaterials. Add a small protein to the gene that we would like to amplify becuase p8 is synthesized so many times- see if this method works and if yes, what applications could this be used for? Insert myc (as in III) or another tag to serve as a “hook” for attaching constructs to M13 tag it (perhaps with flourescence) to learn more about phage coat assembly Add a tag that increases the affinity of the virus coat to various elements that could form nano-constructs. Must be careful when modifying since the sequence of VIII is coupled to that of IX. Change size of protein to experiment with the size of coat. If we change the charge of P8, it will affect how the phage interacts with the surroundings. If we want to locate phages, we could also put markers into P8. - various phage displays: Proteins that will aggregate upon addition of a ligand. Upon mixing with a metal could create thicker nanowires than in prof. Belcher original publication. Mixing of various population interacting with each other could also produce waveguides which might be of great use for electronics. Modify so the shape changes by altering the coat formulation, make the phage more streamlined, sleek so increases proliferation speed.
III	Add myc, or other tags. Change residue sequence to create affinity for different materials. phage tail protein (5 copies): possible mechanism for selectivity to only certain bacteria, dealing with P3 connectivity to the TolA protein on bacterial pilus. present larger moledcules (including myc peitope), change its interactions with bacterial surface molecules to influence which phages are able to replicate, change which bacteria the phage is able to interact with Make the proteins bigger so that they can bind to objects easier and so that they can also bind to bigger objects. Adding a mechanism that could control the expression of functional copies could give us much control over the phage DNA. The p3 protein makes contact with the host and is also the last point of contact when leaving the host. Deletion of this protein greatly slows the exit of new phage particles (thus larger strands of phage can be tolerated because p8 is able to replace the p5 complex). Thus, p3 is able to give us another control over the maximum amount of DNA allowable on the phage sequence. Denaturing the g3 site somehow could allow us to test the level of genetic materiall allowable for varying ration of functional p3 copies to non-functional p3 copies on the surface of the phage. Add myc or alternative tag to monitor the time progression of the phage escape from host. Add myc or other tag to monitor how it affects time delay/progression of phage escape from host add myc epitope, or another marker that can be used for experimentation Change amino acid sequence to make p3 bind to other proteins besides TolA (perhaps allowing it to infect more bacteria) Make the sequence longer to see whether this makes the tail longer and more likely to grasp onto an E. coli cell. Could also change the sequence around to find out by what mechanism p3 works to enter and exit the cell. For example: exchanging charged amino acids for neutral, acidic for neutral or basic, etc. This would take a long time, but could be used to adapt M13 to other hosts for various reasons, since it doesn't kill its host. Modify the proteins that bind to the bacteria (and thus initiate the F pilus and infection) so that the bacteriophage canbind to and infect other types of bacteria- examine the varied life cycles that result Insert myc to allow detection with an antibody Modify in such a way that would allow us to directly control the length of the phages that shed from the E. coli host. For instance, we could delay the time at which the p3/p6 cap is added by making p3 expression a function of environmental cues such as ionic strength or pH. We would also have to take the effect this would have on the infection process since p3 is also the protein which binds to the TolA protein on the bacterial pilus. modify end of protein so that it can bind to other cells (and infect other cells) besides E. Coli Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host Change the GTG to ATG Start? We might change P3 such that the phage was only capable of infecting a different host other than E. coli, if the need arose. Deletion: we could create bacteria grain with extending filaments. Those, when covered with metal, or another material could create grains of a macroscale material [like portland cement which has similar molecular structure.] add a tag to monitor phage escaping from host and the new phages budding from the bacterial surface

Part 3: Data summary for p3-modifications you performed in lab

You'll find guidelines for writing here. Additionally, many of the "for next time" assignments can get you started on this part of the portfolio. Including but not limited to:

• Oligonucleotide design, sequence consequences for phage when inserted and sequence data
• Ligation results (table)
• Agarose gel examining candidate clones (figure)
• Western results (figure)
• Plaque assay (figure)
• Short paragraph for each table and figure describing and interpreting what's shown
• One or two sentence summary of your experimental results
• One or two sentence proposal for what you'd do next if we had one more month to spend on this project

Part 4: Mini-business plan for the Registry of Standard Biological Parts

Put yourself 5 years in the future and imagine that the Registry is floundering. Though the number of useful parts has grown through the hard work and dedication of its volunteer workforce in the iGEM program, there is a notable lack of standards:

• around the parts themselves (some work always, some in rare conditions, some not at all)
• around the assembly process (alternative biobricks and registries have gained popularity)
• and around documentation for the parts (some have great spec sheets and some have nothing).

Decide that you will direct the Registry into a manufacturing, service, high tech, or retail business and then devise a plan to grow and stabilize that business.

In no more than three pages provide a business plan that includes:
1. An Executive Summary In 250 words or fewer, explain:

• • what is your product
  • who are your customers
  • what the future holds for the registry in particular and synthetic biololgy more generally.
  • what you see as the key to success
• This summary should sound enthusiastic, professional and be more readable than most "mission statements."
• consider writing this section after you've written the rest of the plan.
  

2. Summary of the current Registry

• describe what the Registry is, including products, services, customers, ownership, history, location, facilities.
• include strengths and core competencies of the Registry.
• segue into the next section by mentioning the significant challenges faced in the near and long term.
• this section should be no longer than 2 paragraphs.

3. Market analysis Dedicate one paragraph to a description of the market. You might consider including information like:

• who makes up your market?
• what is it's size now? how fast is it growing? how do you know?
• what percentage of the market do you expect the Registry to have now and 5 years from now?
• how could changes in technology, government, and the economy affect your business?
  

4. Business plan Specify your strategy for continued growth of the Registry. The emphasis of this section will differ depending on the kind of business model you have chosen (retail, manufacturing, service or high tech).
Here are some questions you might consider as you formulate your business plan:

• how will you promote the use of the Registry?
• how will you advertise?
• how will you price your product/services.
• where will you locate the Registry (or BioBrick franchises) and how you will distribute parts/services?
• how you will keep the Registry competitive?
• how/if you will protect intellectual property while also promoting sharing and community?
• does your plan emphasize increased production, diversification, or eventual sale of franchises?
• how long will your strategy take to be partially or fully realized?
• are there start-up costs associated with your business model? how much and where will the capital come from?
• will your registry require insurance coverage or litigation insurance?
• are there trademarks, copyrights, or patents (pending, existing, or purchased) considerations?
• how many and what kind (skilled, unskilled, and professional) of employees to you anticipate?
• where will you recruit employees?
• will top notch employees advance? to what?
• how will you training employees?
• what kind of inventory will you keep: raw materials, supplies, finished goods?
• will there be seasonal fluctuations to demand for parts?
• will you need lead-time for ordering?
• do you expect shortages or delivery problems?
• are supply costs steady? reliable?
• will you sell parts on credit?
• how will you set prices?
• what kind of guarantees and privacy protects will you offer?

This section has no defined length or format but should end on an enthusiastic note that might lead some venture capital firm or a funding agency to stay interested.