Difference between revisions of "20.109(F07): Genome engineering assessment"

Latest revision as of 15:54, 15 June 2015

20.109(F07): Laboratory Fundamentals of Biological Engineering

Home People Schedule Fall 2007 Assignments Lab Basics OWW Basics
Genome Engineering Expression Engineering Biomaterials Engineering

General notes:

email these as .doc
email them to nkuldell, endy, nlerner and astachow AT MIT DOT EDU
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....
we will return Parts 1 and 4 with comments and you will have one week to re-submit these portions of your portfolio for up to 1 letter grade improvement in your score on these sections.

Portfolio components

Part 1: Rebuttal to editorial

This will be written as #For next time| a homework assignment, exchanged with your lab partner for peer review, then submitted to the teaching faculty as part of your portfolio. This portion of the assignment accounts for up to 15% of your grade.

Part 2: M13.1 redesign description and parts

Submit your annotated sequence as well as a human-readable text to describe your refactoring. What do you need to do exactly? First, choose a section of the M13.1 scaffold in which you will do your refactoring work (you should choose what you want to refactor based on what seems interesting or important to you). Do not try to refactor all of M13. Only one section between two unique restriction endonuclease sites. Second, design the sequence of DNA for your refactored section of the M13 genome (NOTE: your DNA sequence must start and end with two of the available unique restriction endonuclease sites across the M13 genome). Third, provide the annotated sequence for your refactored section (follow the color scheme suggested, or a better, clearly defined scheme of your own). Don't do anything fancy. A rich text file, word document, or powerpoint file should be good enough. Fourth, provide a one paragraph description of what the purpose or goal of your refactored section is (e.g., what will it test? or, what new function will it provide?). Also, include a cost estimate for your refactored section ($1.35 per bp) and tell us whether or not you think we should have the section synthesized (GO or NO GO). We will quickly check your designs (regardless of GO/NOGO), follow-up w/ any questions, and then order them for DNA synthesis (if you or we say GO). Because we are trying to do this v. quickly this term, it is very important that your clearly annotate your designed sequence (so that we can check it), and provide a good written paragraph describing what your design goals are (so that we can more readily understand what you are hoping to do).This portion of the assignment accounts for up to 40% of your grade.

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)
VIII	add epitope or other tags, e.g. x-ray sensitive, UV sensitive, flourescent. alter gene so 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. change p8 interactions with p5 to regulate size of phage or influence shape of phage by changing how it assembles into a coat. alter gene so fewer copies of p8 fit on phage coat allowing a more flexible packing structure. insert sequence between genes for p5 and p8 to isolate production of these proteins and control their expression levels via activation or repression sites. change amino acid sequence to allow p8 (and thus the entire virus) to bind to certain materials, like metals add a small protein to the gene for p8 to amplify because p8 is synthesized so many times uncoupled gene for p8 from the gene for p9.
III	add epitope or other tags, e.g. x-ray sensitive, UV sensitive, flourescent. modify portion of p3 that normally interacts with TolA on bacterial pilus to vary phage selectivity to different or only certain bacteria. make p3 bigger so that the phage can bind to objects easier and so that they might bind to bigger objects. selective degredation of p3 to control the maximum amount of DNA allowable on the phage sequence. modify to monitor the timing of the phage escape from host. add sequence to make the tail protein longer. make series of changes to explore the mechanism by which p3 enters and exits the cell. For example: exchange charged amino acids for neutral, acidic for neutral or basic, etc. 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. Could also study 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. change the GTG to ATG start.

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:

Table: Oligonucleotide design, sequence consequences for phage when inserted and sequence data
Table: Ligation results
Figure: Agarose gel examining candidate clones
Figure: Western results
Table: Plaque assay
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

This portion of the assignment accounts for up to 20% of your grade.

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. This portion of the assignment accounts for up to 25% of your grade.

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. A helpful link on persuasive writing techniques is here.

@@ Line 1: / Line 1: @@
 {{Template:20.109(F07)}}
-==Portfolio components==
+==<b>General notes:</b>==
-<b>General notes:</b>
 # email these as .doc
 # email them to nkuldell, endy, nlerner and astachow AT MIT DOT EDU
 # 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....
+# we will return Parts 1 and 4 with comments and you will have one week to re-submit these portions of your portfolio for up to 1 letter grade improvement in your score on these sections.
+==Portfolio components==
 ===Part 1: Rebuttal to editorial===
-This will be written as [[20.109(F07): Examine candidate clones| a homework assignment]], exchanged with your lab partner for peer review, then submitted to the teaching faculty as part of your portfolio
+This will be written as [http://openwetware.org/wiki/20.109(F07):_Examine_candidate_clones #For next time| a homework assignment], exchanged with your lab partner for peer review, then submitted to the teaching faculty as part of your portfolio. <b>This portion of the assignment accounts for up to 15% of your grade. </b>
 ===Part 2: M13.1 redesign description and parts===
+Submit your annotated sequence as well as a human-readable text to describe your refactoring. What do you need to do exactly?  First, choose a section of the M13.1 scaffold in which you will do your refactoring work (you should choose what you want to refactor based on what seems interesting or important to you).  Do not try to refactor all of M13.  Only one section between two unique restriction endonuclease sites.  Second, design the sequence of DNA for your refactored section of the M13 genome (NOTE: your DNA sequence must start and end with two of the available unique restriction endonuclease sites across the M13 genome).  Third, provide the annotated sequence for your refactored section (follow the color scheme suggested, or a better, clearly defined scheme of your own).  Don't do anything fancy.  A rich text file, word document, or powerpoint file should be good enough.  Fourth, provide a one paragraph description of what the purpose or goal of your refactored section is (e.g., what will it test? or, what new function will it provide?).  Also, include a cost estimate for your refactored section ($1.35 per bp) and tell us whether or not you think we should have the section synthesized (GO or NO GO).  We will quickly check your designs (regardless of GO/NOGO), follow-up w/ any questions, and then order them for DNA synthesis (if you or we say GO).  Because we are trying to do this v. quickly this term, it is very important that your clearly annotate your designed sequence (so that we can check it), and provide a good written paragraph describing what your design goals are (so that we can more readily understand what you are hoping to do).<b>This portion of the assignment accounts for up to 40% of your grade. </b>
 ====Redesign Ideas from 20.109(S07)====
 {| Border="1"
@@ Line 14: / Line 19: @@
 |-
 | II || <br>
-*Make p2 sensitive to various stimuli (e.g. heat, light) so replication can be regulated.<br>
+* 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.<br>
-*replication of DNA + strand : encode so that P2 can be switched on and off based on the environment which it's in, example: stops replication when in a certain concentration of Ca2+<br>
+* 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<br>
-*make it snesitve to a machansims that can control the proliferation of the phage, perhaps make it require a cofactor that must be added before replication begins<br>
+* make p2 require a cofactor that must be added before replication of the phage begins<br>
-* Keep track of the number of proteins that it nicks.<br>
+* make p2 count the number of times it nicks DNA <br>
-* 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.<br>
+* modify p2 in a way that helps p5 sequester the + strands more effectively, perhaps making p5 work more efficiently by reducing competition. <br>
-* 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.<br>
+* 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). <br>
-* 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.<br>
+* 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<br>
-* add some kind of control to deactivate protein (e.g. sensitivity to heat or a chemical stimuli), this allows better control of the phages<br>
+* alter the gene to make it more active and thus replicate DNA more frequently- see how increased DNA production affects phage growth <br>
-* 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<br>
+* 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<br>
-* 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.<br>
+* remove p2 and p5 to see if the bacteria still make the phage gene products without replicating the phage DNA <br>
-* Alter the gene to make it more active and thus replicate DNA more frequently- see how increased DNA production affects phage growth <br>
-* Modify residues to allow deactivation of p2 under certain conditions so that replication of + strand can be regulated<br>
-* 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.<br>
-* 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<br>
-* Increase or decrease expression to optimize rate of + strand replication.<br>
-*Extract gene X.<br>
-*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.<br>
-* 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.<br>
-* 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 || <br>
-*Make p10 sensitive to a different stimulus than p2 to again regulate replication.<br>
+* make p10 sensitive to a different stimulus than p2 to again regulate replication.<br>
-*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.<br>
+* perhaps a dual control mechanism for p2 and p10 expression<br>
-*changes to II will cause changes to X, perhaps a dual control mechanism<br>
+* increase the number of p10 so that the phage can produce more double strands.<br>
-*Increase the number of proteins so that the phage can produce more double strands.<br>
+* 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.<br>
-* 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.<br>
+* 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. <br>
-* 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).<br>
+* 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.<br>
-* add another way to control phage propagation<br>
-* 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.<br>
-* 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<br>
-* Add sensitivity to different stimulus than that of p2 in order to regulate replication of + strand in another fashion<br>
-* 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.<br>
 * make pX more active so that more + strands will accumulate, allowing the host cell to produce even more phages.<br>
-* Increase expression to see if more phage leave the host E. coli.<br>
+* extract from gene II.<br>
-*Extract from gene II.<br>
-*If we alter p10, we will affect p2. If we don't want phage DNA replication to occur, we can alter this gene.<br>
-* 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.<br>
-* 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 || <br>
-*Add a tag different from p8 (e.g. RFP) to determine what stage of the phage life cycle it is in.<br>
+* 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.<br>
-*binds ssDNA : alter interaction with P9/P7 so that a limited number of ssDNA maybe surrounded by P8 at a time.<br>
+* alter interaction with p9/p7 so that a limited number of ssDNA may be surrounded by p8 at a time.<br>
-*make it sensitive to mechanism that allows for assay of DNA amount and loacatin, change its assembly mechanism to influence phage size<br>
+* make it sensitive to mechanism that allows for assay of DNA amount and location, change its assembly mechanism to influence phage size<br>
-* Modify protein so that similar proteins, other than pVIII can bind to the surface.<br>
+* modify protein so that similar proteins, other than p8 can bind to the surface.<br>
-* 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.<br>
+* 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.<br>
-* Modify such that p5 can sequester the + stranded DNA more effectively so that there is less competition with the formation of double stranded <br>
+* modify such that p5 can sequester the + stranded DNA more effectively so that there is less competition with the formation of double stranded <br>
-*Modify such that it is able to sequester the positive stranded DNA more effectively, thus reducing the competition with dsDNA formation<br>
+* 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 <br>
-*  add tag to track changes, change structure to allow more DNA to fit in the phage<br>
+* 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).<br>
-* Add GFP or other type of tag to enable rapid measurement of DNA replication (an assay of g2/g10)<br>
-* Modify parts of the genome systematically to find out how the interaction with p8 changes with these changes.<br>
-* 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 <br>
-* Add fluorescent tag to monitor levels of p5-ssDNA complex<br>
-* 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).<br>
 * allow it to sequester double stranded DNA also, then it can be used as a vector for infecting cells with desired DNA fragments.<br>
-*  Add more DNA binding sites to see if more DNA can be packaged into phage.<br>
+* add more DNA binding sites to see if more DNA can be packaged into phage.<br>
-* Add some base pairs between V and VII to allow for a restriction site.<br>
+* add some base pairs between V and VII to allow for a restriction site.<br>
-*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.<br>
-* 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.<br>
-*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 || <br>
-*Like, p6, modify to make p9 more flexible.<br>
+* 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.<br>
-*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.<br>
+* change the way p7 interacts with p9<br>
-*change the way it interacts to influence IX's binding affinities<br>
+* modify to increase rate host will shed phage, i.e. decrease the phage-host interaction time.<br>
-* Make is bind more tightly to p9 to allow for more flexibility.<br>
+* modify the last few codons to remove overlap with the gene encoding p9.<br>
-*Chains with desired functions could be attached to pVII. This would place the chains on the exterior of the surface of the phage.<br>
+* add sequence to the 5' end of gene so protein could build nanowires or long filaments or other useful materials. <br>
-* Since p7 is the companion protein of p9, make same modifications as that of p9.<br>
-* Modify in such a way to allow for phage secretion speed to increase so the phage-host interaction time is decreased.<br>
-* make protein 9 more flexible to modifications<br>
-* Change sequence to allow for easier modification of p9 <br>
-* Modify the last few codons so that there is no overlap with g9.<br>
-* Alter the gene so that p8 cannot be substituted for p5- see how this affects the phage (e.g. can it still be secreted?)<br>
-* Minimize the bulk of the protein to allow more room for modifications on p9<br>
-* 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.<br>
 * delete to learn more about its function.<br>
-* Tag protein to monitor interaction with p5/DNA complex.<br>
+* increase it's expression to learn more about its function.<br>
-*Separate from gene IX.<br>
+* tag protein to monitor interaction with p5/DNA complex.<br>
-*Is P7 needed? We could try increasing its expression and/or removing it if we wanted to test this.<br>
-*companion protein to p9, make sure or modify p7 so it can better the interaction with p9, better binding affinity.
 |-
 | IX || <br>
-*Modify p9 to bind to p3 to create long filaments of phage lined up end to end.<br>
+* modify p9 to bind to p3 to create long filaments of phage lined up end to end.<br>
-*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.<br>
+* modify to express different reactive chains on the phage surface.<br>
-*present larger molecules, influence the way DNA is packaged and perhaps thereby controlling proliferation<br>
+* change the function so that p9 will now lyse the bacteria.<br>
-* Change the function so that it now lyse the bacteria.<br>
+* modify to make the phage secretion occur at a faster rate so that interaction time with the host is reduced.
-* 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.<br>
+* modify beginning and end sequences so that g9 does not overlap with g7 and g8.<br>
-* 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.<br>
+* 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)<br>
-*Same principle as gene VII except that care must be taken with this modification because it overlaps with gene 8.<br>
-*  tag, allow p9 to attach to different kinds of proteins including p3 (makes virus chains)<br>
-* Add or change residues to enable p9 to bind to p3, creating long chains of connected filaments like a nanowire<br>
-* Modify beginning and end sequences so that g9 does not overlap with g7 and g8.<br>
-* 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)<br>
-* Add residues to N-terminus to present on the outside of the phage coat<br>
-* 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.<br>
-* modify so that it can act similarly to pIII<br>
-* Tag protein to monitor interaction with p5/DNA complex.<br>
-*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<br>
-* 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.<br>
-*Modify resulting in faster secretion of DNA
 |-
 | VIII || <br>
-*Add myc, or other tags, e.g. GFP.<br>
+* add epitope or other tags, e.g. x-ray sensitive, UV sensitive, flourescent.<br>
-*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.<br>
+* alter gene so 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.<br>
-*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<br>
+* change p8 interactions with p5 to regulate size of phage or influence shape of phage by changing how it assembles into a coat.<br>
-*Make fewer copies so that the proteins can be more flexible.<br>
+* alter gene so fewer copies of p8 fit on phage coat allowing a more flexible packing structure. <br>
-*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.<br>
+* insert sequence between genes for p5 and p8 to isolate production of these proteins and control their expression levels via activation or repression sites. <br>
-* Add myc or alternative tag to aid in targeting various types of hosts. <br>
+* change amino acid sequence to allow p8 (and thus the entire virus) to bind to certain materials, like metals<br>
-* Add myc or alternative tag to possibly aid in targeting various qualities of host<br>
+* add a small protein to the gene for p8 to amplify because p8 is synthesized so many times<br>
-* add myc or another tag (x-ray sensitive, UV sensitive, flourescent)<br>
+* uncoupled gene for p8 from the gene for p9.<br>
-* Change amino acid sequence to allow g8 (and thus the entire virus) to bind to certain materials, like metals<br>
-* 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. <br>
-* 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?<br>
-* Insert myc (as in III) or another tag to serve as a “hook” for attaching constructs to M13<br>
-* 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.<br>
-* Change size of protein to experiment with the size of coat.<br>
-*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.<br>
-*- 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. <br>
-* Modify so the shape changes by altering the coat formulation, make the phage more streamlined, sleek so increases proliferation speed.
 |-
 | III || <br>
-*Add myc, or other tags.  Change residue sequence to create affinity for different materials.<br>
+* add epitope or other tags, e.g. x-ray sensitive, UV sensitive, flourescent.<br>
-*phage tail protein (5 copies): possible mechanism for selectivity to only certain bacteria, dealing with P3 connectivity to the TolA protein on bacterial pilus.<br>
+* modify portion of p3 that normally interacts with TolA on bacterial pilus to vary phage selectivity to different or only certain bacteria. <br>
-*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<br>
+* make p3 bigger so that the phage can bind to objects easier and so that they might bind to bigger objects.<br>
-* Make the proteins bigger so that they can bind to objects easier and so that they can also bind to bigger objects.<br>
+* selective degredation of p3 to control the maximum amount of DNA allowable on the phage sequence. <br>
-* 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.<br>
+* modify to monitor the timing of the phage escape from host.<br>
-* Add myc or alternative tag to monitor the time progression of the phage escape from host.<br>
+* add sequence to make the tail protein longer.
-*  Add myc or other tag to monitor how it affects time delay/progression of phage escape from host<br>
+* make series of changes to explore the mechanism by which p3 enters and exits the cell. For example: exchange charged amino acids for neutral, acidic for neutral or basic, etc. <br>
-* add myc epitope, or another marker that can be used for experimentation<br>
+* 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. Could also study 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.<br>
-* Change amino acid sequence to make p3 bind to other proteins besides TolA (perhaps allowing it to infect more bacteria)<br>
+* change the GTG to ATG start. <br>
-* 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.<br>
-* 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<br>
-* Insert myc to allow detection with an antibody<br>
-* 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.<br>
-* modify end of protein so that it can bind to other cells (and infect other cells) besides E. Coli <br>
-* Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host<br>
-*Change the GTG to ATG Start?<br>
-*We might change P3 such that the phage was only capable of infecting a different host other than E. coli, if the need arose.<br>
-*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.]<br>
-*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 [[20.109(F07):Guidelines for writing a lab report| 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
+*Table: Oligonucleotide design, sequence consequences for phage when inserted and sequence data
-*Ligation results (table)
+*Table: Ligation results
-*Agarose gel examining candidate clones (figure)
+*Figure: Agarose gel examining candidate clones
-*Western results (figure)
+*Figure: Western results
-*Plaque assay (figure)
+*Table: Plaque assay
 *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
+<b>This portion of the assignment accounts for up to 20% of your grade. </b>
 ===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:
@@ Line 176: / Line 111: @@
 *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.
+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. <b>This portion of the assignment accounts for up to 25% of your grade. </b>
 In no more than three pages provide a business plan that includes:  <br>
@@ Line 223: / Line 158: @@
 * 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.
+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. A helpful link on persuasive writing techniques is [http://grammar.ccc.commnet.edu/grammar/composition/argument.htm here.]

Difference between revisions of "20.109(F07): Genome engineering assessment"

Latest revision as of 15:54, 15 June 2015

Contents

General notes:

Portfolio components

Part 1: Rebuttal to editorial

Part 2: M13.1 redesign description and parts

Redesign Ideas from 20.109(S07)

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

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

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