Talk:20.109(S07): DNA ligation and bacterial transformation

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Redesign Ideas from M1D2 FNT assignment

Gene Ideas
I
  • Modify complex such that proteins other than p8 may assemble onto the coat.
  • assembly : alter gene so that surface receptors are present on P1 so that it can determine the number of bacteria phages being secreted at a time.
  • influence how it assembles and interacts with IV to alter size/prperties of channels, this could be important if the size or shape of the phage is changed
  • Modify protein so that similar proteins, other than pVIII can bind to the surface.
  • Attach a well-studied promoter sequence that has known activators and superactivators (or similarly repressors) to the G1 site in order to control transport of new phage out of the host. (This would be done best with a similar modification to XI).
  • Modify so that p1 can interact with multiple p4. Then analyze how phage secretion changes. Changing p1-p4 interaction affects how effective the channels are.
  • Modify such that it is able to interact with multiple p4s and see how phage secretion changes (this would affect the effectiveness of the channels).
  • modify genes I, IV and XI to allow other useful proteins to attach to the complex (more flexibility in phage modification
  • Increase size but maintain shape and interactions, to attempt to create a larger "barrel" to make a wider filament of M13
  • After another copy of g11 is made, edit the ATG sequence that starts g11 so that g1 and g11 can be modified independently. Once this has been done, modify parts of g11 while keeping g1 intact and vice versa to see how this affects replication.
  • Modifiy the gene so that the channels becomes less selective and thus allow other ions, molecules, and proteins to enter and exit the bacteria- see how this affects the life cycles of both the bacteria and the phage
  • Change # of protein copies expressed to experiment with different sized channels
  • Gene I possesses a promoter (Edens et al, 1978). Replace promoter with promoters of known genes that giving various levels of expression (constitutive, uninducible, inducible upon certain environmental conditions) in order to control the amount of membrane channel formation. This would allow us to control the amount of phage secretion (related to the rate of phage infection) from the host.
  • modify gene to that multiple channels can be made, and thus mature phage secretion is accelerated
  • Change size of protein to see effect of different channel sizes.
  • Put genes I, IV, XI under a stronger promoter to create more pores for phage to be extruded though too many pores could be harmful to host cell
  • Separate from genes IV and XI.
  • p1 and p11 interact with p4 so within the bacterial inner membrane, modify p1 to make secretion of the phages more effective. (speed/size of channel)
II
  • Make p2 sensitive to various stimuli (e.g. heat, light) so replication can be regulated.
  • 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+
  • 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
  • Keep track of the number of proteins that it nicks.
  • 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
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
IV
  • Modify complex such that proteins other than p8 may assemble onto the coat.
  • assembly : [look into how P3/P6 cap put on] re-engineer so that multi-phage bacterial links are formed, which requires altering the mechanism of how P4 interacts with the P3/P6 cap with which the phage is secreted.
  • influence its assembly and interaction with I/XI to alter size/properties of channels
  • Modify protein so that similar proteins, other than pVIII can bind to the surface.
  • The n-terminus is believed to interact with the C-terminal ends of pI and pXI. The n-terminal end of pIV could be altered to become an on/off switch to control secretion of phage particles in the presence of an activator or denaturant.
  • Modify in a way that increases p4 affinity for p1. This makes the channels more effective.
  • modify to increase its affinity for p1 and p11--this would possibly increase the effectiveness of the channels
  • After g11 has been removed, decouple from g1 to better manipulate g4. While doing so, keep various lengths of the sequence that was originally before g4 intact to find out if there is a promoter before g4 and, if there is, how long it is.
  • Alter the gene in such a way as to destabalize the outer membrane (e.g. no longer detergent-resistant)- test varying environments for phage survival rate
  • Change # of protein copies expressed to experiment with different sized channels
  • Mutate to make certain strains of M13 that are sensitive to detergent and can only grow in detergent-free environments. Likewise, make certain strains of M13 that are not only resistant to detergent, but also to other potentially harmful substances in the environment. Must be sure not to mutate the end of the gene because that would affect gene I.
  • modify gene to that multiple channels can be made, and thus mature phage secretion is accelerated
  • Change size of protein to see effect of different channel sizes
  • Separate from genes I and XI.
  • Unless phages bottle-neck at the secretion complexes such that secretion becomes the limiting factor in phage production, it does not seem that modifying the proteins that form this complex will help expedite or even really alter our process
  • modify p4 so assembly speed is increased in the outer membrane and modify it so it has a better interaction with p1. This will cause the channels where the phages are secreted to be more effective
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
VI
  • Modify p8 such that it binds to p3 in a way to allow more flexibility in modifying p3.
  • phage tail protein (5 copies): re-engineering closely linked with that of Gene III. Alter it to assist with bacterial selectivity.
  • change the way it interacts to influence III's binding affinities
  • Make it bind more tightly to p3, so that p3 can be modified easily and so that p3 is much more flexible.
  • p6 is a less exposed tail protein. p6 could be altered by inserting a sequence (perhaps using a myc sequence) that would allow it to be tagged with a modified antibody carrying a specific charge. The affects of a charged end could then be studied without disrupting p3.
  • Since p6 is the accessory protein to p3, it can be modified so that p3 can interact more effectively with the host.
  • As an accessory protein to p3, modify such that p3 is more effective in its interaction with TolA protein
  • bind it tighter to phage body, modify protein to allow more modifications to p3
  • Change sequence to allow for easier modification of p3
  • Edit the length of the protein and see what happens. (Does it bulk up under p8/p3, or does it leak out?)
  • Add some sort of tag to the gene that is only visible when p6 is outside of the bacteria- thus we would be able to determine when the phage has been secreted
  • Modify residues to help p3 bind more effectively to the ToIA protein on the E. coli F pilus
  • Control the frequency at which it binds to p3, thus controlling the efficiency of p3. (See III for more details on what ramifications controlling p3 would have.)
  • delete to learn more about its function.
  • Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host.
  • Add some base pairs between III and VI and VI anda I to allow for restriction sites.
  • Is P6 needed? We could test this by increasing the production of P6 and/or by removing the gene for P6. What if we made P6 responsible for either the entrance or final escape from the host, such the P3 only carried on one of these tasks?
  • similar to p3, p6 is the accessory protein to p3. Modify it so it has better and more effective interaction with 3, no interference
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.
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.
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
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
XI
  • Modify complex such that proteins other than p8 may assemble onto the coat.
  • assembly : functionality of 11 is closely linked to that of Gene 4 and Gene 1. Re-engineer so that it can assist with forming multi-phage chains, or so that it can assist with controlling replication.
  • changes to I's interactions with IV will automatically change XI
  • Modify protein so that similar proteins, other than pVIII can bind to the surface.
  • 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
  • Make another copy of g11, then insert it in an area where there is no coding sequence.
  • Modify the gene so that it is longer, hopefully resulting in a larger channel- see if this could allow multiple phages to pass through, thus making the channels more effective
  • Modify residues to allow proteins other than p8 to embed in the membrane and serve as the phage filament coat
  • Can make modifications of similar effect as I (playing around with different levels of expression to regulate membrane channels.)
  • modify gene to that multiple channels can be made, and thus mature phage secretion is accelerated
  • Change size of protein to see effect of different channel sizes.
  • Separate from genes I and IV.
  • modify similar to p1 to change channel properties to make secretion of phages more effective
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