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| | {{Template:TUDelftiGEM2009}} | | {{Template:TUDelftiGEM2009}} |
| | <br> | | <br> |
| - | =Research Proposal=
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| - | __TOC__
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| - | ==Part 1: delay device==
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| - | Time delay genetic circuit
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| - | This kind of circuit has the ability to integrate signals and trigger events after a delay from the initial detection event. There are two approaches to construct a time delay genetic circuit, these are:
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| - | #“protein-based” transcriptional regulators
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| - | #“RNA-based” posttranscriptional regulators<html><sup id="cite_ref-0" class="reference"><a href="#cite_note-0"><span>[</span>1<span>]</span></a></sup></html>
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| - | Based on different literature, we have four different configurations for the time delay genetic circuit. The first two are protein-based and the last two RNA-based.
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| - | ===Negative Feedforward===
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| - | Based on Hooshangi et al.<html><sup id="cite_ref-1" class="reference"><a href="#cite_note-1"><span>[</span>2<span>]</span></a></sup></html> and the project requirements, the next scheme can be proposed.
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| - | <br>[[Image:Negative_feedback.jpg]]<br>
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| - | Figure 1: Genes 1 and 3 are present in the cell and they are produced all the time (black). Genes 2 and 4 (green) are in the self destructive plasmid (SDP). [[Image:Activation_symbol.jpg]] indicates activation and [[Image:Inhibition_symbol.jpg]] indicates repression.<br>
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| - | '''''Description'''''<br>
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| - | Protein 1 starts the circuit activating (or repressing the repressor of) the promoter of gene 2, for proof of concept gene 1 can be changed for a signal molecule. The concentration of protein 2 will increase and achieve the threshold concentration to repress gene 3. At this point the production of protein 3 stops. As the concentration of protein 3 decreases due to its degradation, the repression of gene 4 will not longer exist and the production of protein 4 (restriction enzyme or fluorescent protein) starts.<br>
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| - | '''''Parameters'''''<br>
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| - | In order to make a longer or shorter delay the protein production and degradation rates have to be considered. For the former, we can “play” with the ribosome binding site (RBS) and promoter strength. The degradation can be controlled by protein stability, induction of degradation and up-down regulation of degradation enzymes.<br>
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| - | '''''Biobricks'''''<br>
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| - | #new RBS
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| - | #new transcriptional factors with different stabilities
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| - | #new promoters with different strength<br>
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| - | '''''Take into account'''''<br>
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| - | *There are many of biobricks 1 and 3 in the registry
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| - | *Time frame
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| - | *Change LVA tag to decrease degradation<br>
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| - | '''''Modeling'''''<br>
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| - | Differential equations involving production rate, degradation rate (and dilution rate)
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| - | ===Positive feedforward===
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| - | Based on the book of Uri Alon<html><sup id="cite_ref-2" class="reference"><a href="#cite_note-2"><span>[</span>3<span>]</span></a></sup></html> and the project requirements, the next scheme can be proposed.
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| - | <br>[[Image:Positive_feedforward.jpg]]<br>
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| - | Figure 2: Genes 1 and 3 are present in the cell and they are produced all the time (black). Genes 2 and 4 (green) are in the self destructive plasmid (SDP).<br>
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| - | '''''Description'''''<br>
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| - | Protein 1 starts the circuit activating the promoter of gene 2, for proof of concept gene 1 can be changed for a signal molecule. The concentration of protein 2 will increase and achieve the threshold concentration to induce gene 3. As the concentration of protein 3 increases, the induction of gene 4 (restriction enzyme or fluorescent protein) starts.<br>
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| - | '''''Parameters'''''<br>
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| - | In order to make a longer or shorter delay the protein production and degradation rates have to be considered. For the former, we can “play” with the ribosome binding site (RBS) and promoter strength. The degradation can be controlled by protein stability, induction of degradation and up-down regulation of degradation enzymes.<br>
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| - | '''''Biobricks'''''<br>
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| - | #new RBS
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| - | #new transcriptional factors with different stabilities
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| - | #new promoters with different strength<br>
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| - | '''''Take into account'''''<br>
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| - | *There are many of biobricks like 1 and 3 in the registry
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| - | *Positive feed is always related to leaking problems
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| - | *Time frame
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| - | *Change LVA tag to decrease degradation<br>
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| - | '''''Modeling'''''<br>
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| - | Differential equations involving production rate, degradation rate (and dilution rate)
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| - | ===Riboregulator===
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| - | In bacteria, several factors affect translation initiation, including ribosomal recognition of the mRNA’s RBS and the start codon (AUG). Recognizing the importance of RNA interactions between the ribosome and RBS, and based on work on endogenous riboregulators, Isaacs et al.<html><sup id="cite_ref-3" class="reference"><a href="#cite_note-3"><span>[</span>4<span>]</span></a></sup></html> sought to regulate bacterial gene expression by interfering with ribosomal docking at the RBS. From the outset, their objective was to create a modular post transcriptional regulation system that could be integrated into biological networks and implemented with any promoter or gene. This system was already used by Friedland et al<html><sup id="cite_ref-4" class="reference"><a href="#cite_note-4"><span>[</span>5<span>]</span></a></sup></html>.
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| - | Circuit 3 that we have in mind is based on the riboregulator and is shown in figure 3.
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| - | <br>[[Image:Riboregulator.jpg]]<br>
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| - | Figure 3: taRNA/RBS (riboregulator) regulated expression of protein 3. Genes 1 and 2 are on the conjugative plasmid present in all cells. S1: signal molecule. Gene 3 is present on the SDP. RBS: ribosome binding site. Cr: piece of RNA strand in front of RBS that is complementary to RBS. taRNA: trans-activating RNA, could also be small interference RNA, as long as it is complementary to cr. Product 3: Could be either GFP/luciferase (for testing) or endonuclease.<br>
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| - | '''''Description'''''<br>
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| - | Either gene 1 or gene 2 is constitutively expressed (never both!) where the other is inducible. In the picture, gene 2 is constitutive and gene 1 is inducible by signal molecule S1. Gene 2 expresses constitutively the crRNA, on which the cr part of the RNA binds the RBS directly after transcription, causing RBS to be blocked for the ribosome and therefore blocks the RNA from translation. When the promoter of gene 1 is induced by S1, it produces the taRNA molecule which is complementary to the cr part of the crRNA. When the taRNA binds the crRNA it opens the RBS, which makes the crRNA ready for translation. The resulting product (the green folded protein 2) can then induce gene 3 to produce end-product 3.<br>
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| - | '''''Parameters and take into account'''''
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| - | #taRNA should be better complementary to cr than cr to RBS, to make sure that taRNA causes the RBS to open up.
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| - | #One can play with promoter strengths for genes 1 and 3 (or when gene 1 is constitutively expressed, genes 2 and 3).
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| - | #The degradation rate of protein 2 (the green folded protein in figure 3) can influence the delay time.
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| - | #Instead of protein 2 inducing gene 3 expression, it might repress a repressor of promoter 3 (negative feed-forward, see figure 1), to get rid of leaky expression.
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| - | #Take into account “scars”
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| - | #Time frame<br>
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| - | '''''Biobricks'''''<br>
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| - | #new RBS
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| - | #new transcriptional factors with different stabilities
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| - | #new promoters with different strength
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| - | #crRNA for each RBS in the registry
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| - | #taRNA for each crRNA<br>
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| - | '''''Modeling'''''<br>
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| - | *Differential equations involving production rate, degradation rate (and dilution rate)
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| - | *Interaction crRNA’s – RBS and crRNA’s – taRNA’s
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| - | *Stability of taRNA’s
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| - | ===Small interfering RNA===
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| - | This approach is based on the gene silencing by small interfering RNA explained in Elbashir et al.<html><sup id="cite_ref-5" class="reference"><a href="#cite_note-5"><span>[</span>6<span>]</span></a></sup></html> and is given in the following scheme.
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| - | <br>[[Image:small_interfering_RNA.jpg]]<br>
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| - | Figure 4: Genes 1 and 3 are present in the self destructive plasmid. Gene 2 is present in the cell and is always produced.<br>
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| - | '''''Description'''''<br>
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| - | Protein 1 is always produced in the cell and is controlled by the constitutive promoter on gene 2. This protein must be selected carefully since the gene 1 producing the siRNA must be decided based on this protein and the siRNA must be specific for silencing the mRNA of protein 1 without disturbing the other mRNAs of the host organism. The protein 1 will be in excess concentration and hence will inhibit the production of protein 2 (restriction enzyme or fluorescence protein) by repressing the promoter of gene 3. When the production of siRNA is induced by activating the promoter of gene 1, on reaching a threshold it will interfere with the translation of protein 1 and hence the repression of gene 3 is released to produce the protein 2.<br>
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| - | '''''Parameters and take into account'''''<br>
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| - | *Protein 1’s stability
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| - | *siRNA specificity
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| - | *Promoter strength.<br>
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| - | '''''Biobricks'''''<br>
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| - | #new RBS
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| - | #new transcriptional factors with different stabilities
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| - | #new promoters with different strength
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| - | #siRNA’s<br>
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| - | '''''Modeling'''''<br>
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| - | *Differential equations involving production rate, degradation rate (and dilution rate)
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| - | *Interaction siRNA’s - mRNA
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| - | ===Which biobricks we could use===
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| - | <table class="table1" border="1" style="text-align: center;"><tbody>
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| - | <tr><th>Repressible promoters</th><th>Biobrick</th><th>well/plasmid/plate 2009</th></tr>
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| - | <tr><th>Plac</th><th>R0010</th><th>1D/pSB1A2/1</th></tr>
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| - | <tr><th>Ptet</th><th>R0040</th><th>6I/pSB1A2/1</th></tr>
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| - | <tr><th>λp</th><th>R0051</th><th>6K/pSB1A2/1</th></tr>
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| - | <tr><th>λp 434cI</th><th>R0052</th><th>11D/pSB2K3/2</th></tr>
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| - | <tr><th>p 22 cII</th><th>R0053</th><th>6M/pSB1A2/1</th></tr>
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| - | </tbody></table>
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| - | Repressor molecule Biobrick well/plasmid/plate 2009 More
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| - | Lac I C0012 2O/pSB1A2/1 IPTG induces the promoter blocking
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| - | Lac I, this biobrick has LVA
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| - | Lac IS K142000 --- IPTG does not have any effect
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| - | TetR C0040 4A/pSB1A2/1 aTc induces the promoter blocking
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| - | TetR, this biobrick has LVA
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| - | cI C0051 4E/pSB1A2/1 this biobrick has LVA
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| - | 434 cI C0052 4G/pSB1A2/1 this biobrick has LVA
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| - | 22 cII C0053 4I/pSB1A2/1 this biobrick has LVA
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| - |
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| - | Other promoters Biobrick well/plasmid/plate 2009 More
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| - | PT7 I712074 6N/pSB1AK8/1 Only useful when
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| - | T7 RNAP is present
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| - |
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| - | RNA polymerase Biobrick well/plasmid/plate 2009 More
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| - | T7 RNAP I716103 3A/pSB1A2/2 ATG start codon
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| - |
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| - | Other Biobrick More
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| - | LVA tag M0040 Just planned
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| - |
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| - |
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| - | Available in kit 2009
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| - | ==Part 3: conjugation==
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| - | ===Selecting the Conjugation system===
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| - | There are many different conjugation systems, with F and IncP being among the most used by previous iGEM teams. The IncP incompatibility group is further divided into two groups IncP-alpha aka Birmingham aka RP4 plasmid and the IncP-beta R751 plasmid. There are several reasons why the IncP system would be better suited for us:
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| - | *Smaller plasmid size (R751 is 53423<html><sup id="cite_ref-6" class="reference"><a href="#cite_note-6"><span>[</span>7<span>]</span></a></sup></html>) (RP4 is 60099<html><sup id="cite_ref-7" class="reference"><a href="#cite_note-7"><span>[</span>8<span>]</span></a></sup></html>) (F is 99159<html><sup id="cite_ref-8" class="reference"><a href="#cite_note-8"><span>[</span>9<span>]</span></a></sup></html>)
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| - | *F has a self-imposed fertility inhibition system (new donors fertile for only about 6 generations), IncP systems have no self-imposed fertility inhibition system<html><sup id="cite_ref-9" class="reference"><a href="#cite_note-9"><span>[</span>10<span>]</span></a></sup></html>
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| - | *F has two surface exclusion proteins (TraT, TraS), IncP has only one (trbK)<html><sup id="cite_ref-9" class="reference"><a href="#cite_note-9"><span>[</span>10<span>]</span></a></sup></html>
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| - | ===Selecting genes to knockout===
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| - | '''Entry exclusion'''<br>
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| - | The goal of this part of the project is to allow a message plasmid to be transmitted between cells containing conjugation helper plasmids (modified R751). In nature this occurs but at very low levels, due to the presence of entry exclusion proteins<html><sup id="cite_ref-10" class="reference"><a href="#cite_note-10"><span>[</span>11<span>]</span></a></sup></html>. These membrane-bound proteins block incoming transfers from cells containing the conjugative plasmid. Note that in order for the transfer to be blocked their presence is required only in the recipient. These entry exclusion proteins are present for two primary reasons: i) to prevent redundant transfer of the conjugative plasmid among a population of cells and ii) to prevent lethal zygosis<html><sup id="cite_ref-11" class="reference"><a href="#cite_note-11"><span>[</span>12<span>]</span></a></sup></html>. In R751 the gene trbK encodes for the entry exclusion protein<html><sup id="cite_ref-12" class="reference"><a href="#cite_note-12"><span>[</span>13<span>]</span></a></sup></html> in R751 and RP4. Its presence is not required for conjugation to occur<html><sup id="cite_ref-13" class="reference"><a href="#cite_note-13"><span>[</span>14<span>]</span></a></sup></html>. Some papers have reported that knocking out the trbK genes in other plasmids did not affect the conjugation frequency: “transfer of the trbK mutant occurred at near-wild-type frequencies”<html><sup id="cite_ref-14" class="reference"><a href="#cite_note-14"><span>[</span>15<span>]</span></a></sup></html>. Given this information we propose to knockout the trbK gene.<br>
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| - | '''Lethal Zygosis'''<br>
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| - | What is lethal zygosis: “We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing.<html><sup id="cite_ref-11" class="reference"><a href="#cite_note-11"><span>[</span>12<span>]</span></a></sup></html>”
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| - | By knocking out the trbK gene we may be exposing our cells to lethal zygosis. If this is indeed the case than knocking out one of the critical mating pair formation genes (trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbL) from the trb operon would prevent this. The gene trbC was selected for this due to its small size and position on the operon. trbC is known to encode for the pilin subunits needed for pilus formation. Given this information we propose to knockout the trbC gene as well.
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| - | ===Experimental Procedures===
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| - | '''Section 1: Helper Plasmid'''<br>
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| - | Part 1A:
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| - | *Acquire R751 and/or RP4 plasmid
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| - | *Electoporate plasmid into cells and confirm conjugation. See [http://openwetware.org/wiki/Conjugation conjugation protocol]
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| - | *Characterize conjugation efficiency
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| - | Part 1B: oriT knockout
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| - | *Knockout oriT using either [http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement lambda red], [http://openwetware.org/wiki/IGEM:Peking/2007/Count:Knockout knockout protocol used by Peking '07] or [http://openwetware.org/wiki/Berk2006-ConjugationTeam knockout protocol used by Berkeley '06]
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| - | *Electroporate into cells
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| - | *Verify that conjugation stopped
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| - | *Electroporate PlasmidG as well and characterize conjugation efficiency
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| - | *If works send R751 oriT knockout plasmid to registry
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| - | Part 1C: trbK knockout
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| - | *Knockout oriT + trbK
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| - | *Electroporate into cells
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| - | *Verify that conjugation takes place among R+ cells using PlasmidG (see below)
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| - | *Characterize conjugation efficiency
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| - | *If works send R751 oriT + trbK knockout plasmid to registry
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| - | Part 1D: trbC knockout
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| - | *Knockout oriT + trbK + trbC
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| - | *Electroporate into cells and create culture for communication (cultureCom)
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| - | *Verify that no conjugation takes place in presence of PlasmidG
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| - | *If works send R751 oriT + trbK + trbC knockout plasmid to registry
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| - | '''Section 2: Message Plasmid'''<br>
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| - | Part 2A: BioBrick Assembly
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| - | *Order DNA synthesis for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 BBa_K175001] (trbK)
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| - | *Amplify BioBricks needed [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (GFP generator), [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] (strong rbs), [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (constitutive promoter), [http://partsregistry.org/wiki/index.php?title=Part:BBa_I714031 BBa_I714031] (oriT-R).
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| - | *Assemble: [oriT][promoter] and keep this for later
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| - | *Assemble PlasmidG: [oriT][promoter][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
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| - | *Assemble [oriT][promoter][rbs][trbC][rbs][trbK] and keep this intermediate assembly product for possible use in integration stage
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| - | *Assemble [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] and keep this intermediate assembly product for possible use in integration stage
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| - | *Assemble PlasmidCKG: [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
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| - | Part 2B: Full Communication testing
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| - | *Electroporate PlasmidCKG into some cells from cultureCom creating initiatorCells
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| - | *Select for presence of both message and helper plasmid
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| - | *Mix initiatorCells with cells not containing any plasmids and characterize conjugation efficiency
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| - | *Add initiatorCells to cultureCom and observe signal propagation, characterize rate of signal propagation.
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| - | *If signal propagation observed, do victory dance.
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| - | ='''References'''=
| |
| - | ==Delay device==
| |
| - | <li id="cite_note-0"><cite id="CITEREFSmolke2009" class="article" style="font-style: normal;">
| |
| - | Smolke C, 2009. [http://www.ncbi.nlm.nih.gov/pubmed/19478174 It’s the DNA That Counts]. Science, 324:1156-1157.</cite>
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| - | <li id="cite_note-1"><cite id="CITEREFSmolke2009" class="article" style="font-style: normal;">
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| - | Hooshangi S, Thiberge S, Weiss R, 2005. [http://www.ncbi.nlm.nih.gov/pubmed/15738412 Ultrasensitivity and noise propagation in a synthetic transcriptional cascade]. PNAS, 102,10 :3581–3586.</cite>
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| - | <li id="cite_note-2"><cite id="CITEREFSystemBio2006" class="book" style="font-style: normal;">
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| - | An introduction to systems biology: design principles of biological circuits. Uri Alon, Publisher CRC Press, 2006, pp 41-69.[http://en.wikipedia.org/wiki/Special:BookSources/1584886420 ISBN 1584886420]</cite>
| |
| - | <li id="cite_note-3"><cite id="CITEREFIsaacs2006" class="article" style="font-style: normal;">
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| - | Isaacs F, Dwyer d and Collins J, 2006. [http://www.ncbi.nlm.nih.gov/pubmed/16680139 RNA synthetic biology]. Nature Biotech., 24:545-554.</cite>
| |
| - | <li id="cite_note-4"><cite id="CITEREFFriedland2009" class="article" style="font-style: normal;">
| |
| - | Friedland A, Lu T, Wang X, Shi D, Church G and Collins J, 2009. [http://www.ncbi.nlm.nih.gov/pubmed/19478183 Synthetic Gene Networks That Count]. Science, 324:1199-1202.</cite>
| |
| - | <li id="cite_note-5"><cite id="CITEREFElbashir2001" class="article" style="font-style: normal;">
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| - | Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T., 2001. [http://www.ncbi.nlm.nih.gov/pubmed/11373684 Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells]. Nature, 411(6836):494-498.</cite>
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| - | ==Conjugation==
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| - | <li id="cite_note-6"><cite id="CITEREFThorsted1998" class="article" style="font-style: normal;">
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| - | Thorsted, P. B., D. P. Macartney, P. Akhtar, A. S. Haines, N. Ali, P. Davidson, T. Stafford, M. J. Pocklington, W. Pansegrau, B. M. Wilkins, E. Lanka, and C. M. Thomas. 1998. [http://www.ncbi.nlm.nih.gov/nuccore/113911681?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone]. J. Mol. Biol. 282:969-990.</cite>
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| - | <li id="cite_note-7"><cite id="CITEREFPansegrau1995" class="article" style="font-style: normal;">
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| - | Pansegrau, W., E. Lanka, P. T. Barth, D. H. Figurski, D. G. Guiney, D. Haas, D. R. Helinski, H. Schwab, V. A. Stanisich, and C. M. Thomas. 1995. [http://www.ncbi.nlm.nih.gov/nuccore/508311?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Complete nucleotide sequence of Birmingham IncP alpha plasmids]. Compilation and comparative analysis. J. Mol. Biol. 239:623-663.</cite>
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| - | <li id="cite_note-8"><cite id="CITEREFFrost1995" class="article" style="font-style: normal;">
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| - | Frost, L., K. Ippen-Ihler, and M. Skurray. 1995. [http://www.ncbi.nlm.nih.gov/nuccore/9507713?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Analysis of the sequence and gene products of the transfer region of the F sex factor]. Microbiol. Rev. 58:162-210.</cite>
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| - | <li id="cite_note-9"><cite id="CITEREFPlasBio2004" class="book" style="font-style: normal;">
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| - | Plasmid Biology. Barbara E. Funnell and Gregory J. Phillips, eds. American Society for Microbiology Press, Washington, DC, 2004. Chapter 9.[http://en.wikipedia.org/wiki/Special:BookSources/1555812651 ISBN 1555812651]</cite>
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| - | <li id="cite_note-10"><cite id="CITEREFGarcillán-Barcia2008" class="article" style="font-style: normal;">
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| - | Garcillán-Barcia MP, de la Cruz F. [http://www.ncbi.nlm.nih.gov/pubmed/18440635 Why is entry exclusion an essential feature of conjugative plasmids?] 2008;60:1–18. doi: 10.1016/j.plasmid.2008.03.002.</cite>
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| - | <li id="cite_note-11"><cite id="CITEREFOu1980" class="article" style="font-style: normal;">
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| - | Ou, J. T. 1980. [http://www.ncbi.nlm.nih.gov/pubmed/6993854 Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating]. Molecular and General Genetics 178:573–581.</cite>
| |
| - | <li id="cite_note-12"><cite id="CITEREFHaase1996" class="article" style="font-style: normal;">
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| - | Haase, J., Kalkum, M. & Lanka, E., 1996. [http://www.ncbi.nlm.nih.gov/pubmed/8955288 TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4]. J. Bacteriol. 178, 6720–6729.</cite>
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| - | <li id="cite_note-13"><cite id="CITEREFHaase1995" class="article" style="font-style: normal;">
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| - | Haase J, Lurz R, Grahn A M, Bamford D H, Lanka E., 1995. [http://www.ncbi.nlm.nih.gov/pubmed/7642506 Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex]. J Bacteriol. 177:4779–4791.</cite>
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| - | <li id="cite_note-13"><cite id="CITEREFLi1999" class="article" style="font-style: normal;">
| |
| - | Li, P. L., Hwang, I., Miyagi, H., True, H., Farrand, S. K., 1999. [http://www.ncbi.nlm.nih.gov/pubmed/10438776 Essential components of the Ti plasmid trb system, a type IV macromolecular transporter]. J. Bacteriol. 181:5033-5041.</cite>
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