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| {| border="1" | | {| border="1" |
| ! protein | | ! Diagnostic digest 1 |
| ! function!! Genome Re-Engineering Plans
| | | plasmid with insert |
| | | plasmid no insert |
| |- | | |- |
| | I | | | Enzyme(s) used |
| | assembly | | | ZraI |
| | 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). | | | BamHI |
| |- | | |- |
| | II | | | Buffer used |
| | replication of DNA + strand | | | SEBuffer B NEBuffer 1, 2, 4 |
| | 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. | | | NEBuffer 2 |
| |- | | |- |
| | III | | | Temperature |
| | phage tail protein (5 copies) | | | 37 C |
| | 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. | | | 37 C |
| |- | | |- |
| | IV | | | Predicted fragments |
| | assembly | | | 1 DNA fragment the length of the M13 backbone plus the insert |
| | 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. | | | 1 Fragment the lenght of the original M13 genome |
| |- | | |- |
| | V | | | |
| | binds ssDNA | | | |
| | 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. | | | |
| |- | | |- |
| | VI
| | ! Diagnostic digest 2 |
| | phage tail protein (5 copies) | | | |
| | 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. | | | |
| |- | | |- |
| | VII | | | Enzyme(s) used |
| | phage head protein (5 copies) | | | BamHI |
| |Chains with desired functions could be attached to pVII. This would place the chains on the exterior of the surface of the phage. | | | ZraI |
| |- | | |- |
| | VIII | | | Buffer used |
| | phage coat protein (2700 copies) | | | NEBuffer 2 |
| |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. | | | SEBuffer B; NEBuffer 1,2,4 |
| |- | | |- |
| | IX | | | Temperature |
| | phage head protein (5 copies) | | | 37 C |
| | 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. | | | 37 C |
| |- | | |- |
| | X | | | Predicted fragments |
| | DNA replication | | | No linear fragments. Plasmid remains circular. |
| | see pII modification
| | | No linear fragments. Plasmid remains circular. |
| |-
| |
| | XI
| |
| | assembly
| |
| | Another known promoter sequence could then be added (in conjunct with g1 modification)in order to control levels of p1 and p11. The promoter sequences would allow isolation of protein production in order to study effects of activated or repressed expression levels. | |
| |} | | |} |
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