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 various stimuli (e.g. heat, light) 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 pII shares a commmon C-terminal portion with p10. the gII site could be altered so that this C-terminal portion is modified to possess a sensisitivity to some sort of stimuli such as pH. Transcriptional regulation could be achieved if the modification is able to evoke some sort of change in pII (and pX) proteins, such as denaturation. Modify such that it nicks the DNA in a way that helps p5 sequester the + strands more effectively, thus controlling the formation of double stranded DNA and perhaps making p5 work more efficiently by reducing competition. Be careful in modifying because it is linked to g10. 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 g5). Care should be taken when modifying because this is also linked to g10. add some kind of control to deactivate protein (e.g. sensitivity to heat or a chemical stimuli), this allows better control of the phages 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 After another copy of g10 is made, edit the ATG sequence that starts g10 so that g2 and g10 can be modified independently. Modify parts of g2 or g10 while keeping the other intact to see how the disparate sequences affect replication. Alter the gene to make it more active and thus replicate DNA more frequently- see how increased DNA production affects phage growth Modify residues to allow deactivation of p2 under certain conditions so that replication of + strand can be regulated One way to control rate of phage infection is to control replication of phage DNA based on stimuli from the environment. Assuming gene II also has a promoter, we could implement a strategy similar to that used for gene I. 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 Increase or decrease expression to optimize rate of + strand replication. Extract gene X. Without P2, could the bacteria still make the phage gene products without replicating the phage DNA? If we remove P2 (and P5), could we use this virus to insert other genes into the bacteria? NOTE: Altering the second half of P2 will affect P10. Enclosing the gene II in maltose binding protein: http://www.jhu.edu/~cheme/ostermeier/pdf/19_JMB.pdf by random domain insertion. This would allow to control the rate of synthesis of + strand thus delaying replication until the right moment.This principle could be connected for example to quorum sensing [bacteria producing maltose?], thus creating a kind of bacterial "war" with suicide bombers: whenever a bacteria from another group enters in vicinity with its maltose - the cell releases phages. [I thought it would be a funny application.]. This approach would be probably faster than transcription induction. modify p2 so it nicks in such a way to make p5 respond quicker, send out a signal to p5 so it can protect the + stranded DNA
X	Make p10 sensitive to a different stimulus than p2 to again regulate replication. DNA replication : since altering Gene X is synonymous with altering Gene II, I would leave it alone and concentrate more on changing the functionality of gene 2. This makes more sense since Gene II as a better defined function within m13. changes to II will cause changes to X, perhaps a dual control mechanism Increase the number of proteins 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. Modify such that + strands are not dependent on the presense of p10. This would work in conjuction with our modification of p2 (since they are linked). add another way to control phage propagation Make another copy of g10, then insert it in an area where there is no coding sequence. Put a tag on p10 and see what it binds to at various parts of replication. This will help to elucidate how it controls the amount of double stranded M13 genomes. See g2 entry for other details. Altering this gene will also alter gene 2 so any alteration would affect both genes, so make any number of small modifications - see what interesting phenomena result Add sensitivity to different stimulus than that of p2 in order to regulate replication of + strand in another fashion 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. Increase expression to see if more phage leave the host E. coli. Extract from gene II. If we alter p10, we will affect p2. If we don't want phage DNA replication to occur, we can alter this gene. Similarly as in pIII, but instead of deletion we could change the promoters for more or less efficient. This would allow to control the replication rate. If the pX is infunctional for a longer period - no + strands will accumulate and no replication will occur. Therefore, the smaller concentration should decrease the replication rate. linked to p2, modify so p10 is not so dependent on 2 and the DNA can still replicate and modify so there is better accumulation of + stranded DNA
V	Add a tag different from p8 (e.g. RFP) to determine what stage of the phage life cycle it is in. binds ssDNA : alter interaction with P9/P7 so that a limited number of ssDNA maybe surrounded by P8 at a time. make it sensitive to mechanism that allows for assay of DNA amount and loacatin, change its assembly mechanism to influence phage size Modify protein so that similar proteins, other than pVIII can bind to the surface. pV overlaps with pVII's start codon. An activation site could be added in prior to pVII's start codon 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 Modify such that it is able to sequester the positive stranded DNA more effectively, thus reducing the competition with dsDNA formation add tag to track changes, change structure to allow more DNA to fit in the phage Add GFP or other type of tag to enable rapid measurement of DNA replication (an assay of g2/g10) Modify parts of the genome systematically to find out how the interaction with p8 changes with these changes. Vary the activity of the gene and thus the competition between dsDNA formation and the sequestering of ssDNA- compare the results to find the optimum level of phage production possible Add fluorescent tag to monitor levels of p5-ssDNA complex 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. If we have a stronger promoter in front of this gene, phage packaging might occur faster, giving us more efficient production times. However, if too many + strands are taken away, this will hurt the DNA Replication process. Furthermore, no matter how many + strands are sequestered, we will still need enough coat protein to package these strands of DNA, so a strong promoter in front of this gene alone might prove useless. This protein can bind + ssDNA. The gene should be isolated and we should check whether it can also protect from restriction nucleases, or does it increase stability of DNA. modify p5 so it can better protect the + stranded DNA which will lead to more rapid replication and amplification; less competition with the double stranded DNA
VII	Like, p6, modify to make p9 more flexible. phage head protein (5 copies): alter gene so as to alter its conformation. A change in conformation can expand the different residues that can be attatched to its N-terminal portion. change the way it interacts to influence IX's binding affinities Make is bind more tightly to p9 to allow for more flexibility. Chains with desired functions could be attached to pVII. This would place the chains on the exterior of the surface of the phage. Since p7 is the companion protein of p9, make same modifications as that of p9. Modify in such a way to allow for phage secretion speed to increase so the phage-host interaction time is decreased. make protein 9 more flexible to modifications Change sequence to allow for easier modification of p9 Modify the last few codons so that there is no overlap with g9. Alter the gene so that p8 cannot be substituted for p5- see how this affects the phage (e.g. can it still be secreted?) Minimize the bulk of the protein to allow more room for modifications on p9 Add sequence to code for additional residues to add to the N-terminal end (could form the basis for building nano-wires or long filaments of other useful materials. Must be careful when inserting sequence since VII is coupled to IX. delete to learn more about its function. Tag protein to monitor interaction with p5/DNA complex. Separate from gene IX. Is P7 needed? We could try increasing its expression and/or removing it if we wanted to test this. companion protein to p9, make sure or modify p7 so it can better the interaction with p9, better binding affinity.
IX	Modify p9 to bind to p3 to create long filaments of phage lined up end to end. phage head protein (5 copies): re-engineering closely linked to that of Gene VII. However, since 9 is located on the surface of m13, it can be altered so that it can express different reactive chains on the phage surface. present larger molecules, influence the way DNA is packaged and perhaps thereby controlling proliferation Change the function so that it now lyse the bacteria. p9 exists on the surface of the head. p9 could be tagged with a variety of functional chains desired to be found on the exterior of the phage's protein coat. Modify in such a way to make the phage secretion occur at a faster rate so that interaction time with the host is reduced. Also, modify so that p9 interaction with p5 is more effective. Since part of g9 overlaps with g8, be careful in making modifications. Same principle as gene VII except that care must be taken with this modification because it overlaps with gene 8. tag, allow p9 to attach to different kinds of proteins including p3 (makes virus chains) Add or change residues to enable p9 to bind to p3, creating long chains of connected filaments like a nanowire Modify beginning and end sequences so that g9 does not overlap with g7 and g8. Modify the gene so that it can bind to bacterial surface proteins (like p3 does)- see if this allows the phage to interact with other bacteria (now that both ends can bind) Add residues to N-terminus to present on the outside of the phage coat Could implement an idea similar to the one for VII, since they both can have residues added to their N-terminal end, and are similar in size and function. Must be careful when modifying since the sequence of IX is coupled to that of VII and VIII. modify so that it can act similarly to pIII Tag protein to monitor interaction with p5/DNA complex. 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.