Difference between revisions of "Team:BIT-China/Design"

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        .content-title{
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            text-align: center;
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            font-size: 2.5em;
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        .block-title{
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        .problem-title{
 +
            font-size: 1.5em;
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        }
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        .problem-txt div{
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<div class="col-sm-12" style="padding: 0;background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)">
 
<div class="col-sm-12" style="padding: 0;background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)">
−
     <div class="nav-left" id="nav_left_wrapper"  style="width: 200px;">
+
   
 +
    <div class="content-right" style="float: left;width:100%;padding: 10px;">
 +
        <div style="margin-left:220px;background: white;margin-top: 0.5em;margin-bottom:0.5em;padding: 1px;border-radius: 10px">
 +
            <div class="overview-content" style="border:1px dashed #F08B1F;
 +
            border-radius: 10px;margin: 0.5em;padding: 10px;">
 +
 
 +
                <div class="content-title">Design</div>
 +
 
 +
                <!--问题描述-->
 +
                <div class="problem-txt">
 +
                    <div class="problem-title">What is the problem?</div>
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"
 +
                            alt="ul_logo1" width="18" height="18">
 +
                    <span>Plasmid segragational instability has been the limit
 +
                        step for large-scale protein production and bioremediation.
 +
                        Use of antibiotics for plasmid retention is not practical
 +
                        in this situation.</span>
 +
                    </div>
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"
 +
                            alt="ul_logo1" width="18" height="18">
 +
                    <span>High copy number (hcn) plasmids are lost from population at a high rate due to the
 +
                        large metabolic burden and lack of nature factors for plasmid maintenance.</span>
 +
                    </div>
 +
                </div>
 +
 
 +
                <!--Our P-SLACKiller-->
 +
                <div class="block-title">Our P-SLACKiller</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-item">
 +
                        <!--Our P-Slackiller小分块-->
 +
                        <div class="block-content-item-block">
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                    alt="ul_logo1" width="18" height="18">
 +
                                <span>IDEA</span>
 +
                            </div>
 +
                            <div>
 +
                                Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller). Use the inhibitor protein as a signal indicating plasmid numbers,
 +
                                when the plasmid concentration reduces to a threshold, the switch of killer gene will turn on.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig1.png"
 +
                                    alt="fig1" class="center-block" style="width: 60%;">
 +
                            </div>
 +
                        </div>
 +
 
 +
                        <!--小分块-->
 +
                        <div class="block-content-item-block">
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                    alt="ul_logo1" width="18" height="18">
 +
                                <span>PART DESIGN</span>
 +
                            </div>
 +
                            <div>
 +
                                The basic circuit is shown in Fig.2, the genetic locus of functional gene can be substituted on the basis of production demand in the future. Inhibitor and in-promoter are key to control the threshold.
 +
                                When the signal of plasmid losing is detected, the executor killer will play a role.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/0/01/T--BIT-China--Project--Design--fig2.png"
 +
                                    alt="fig2" class="center-block" style="width: 60%;">
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
 
 +
                <!--Killer device-->
 +
                <div class="block-title">Killer device</div>
 +
                <div class="block-content">
 +
                    <!--Killer Device 简介-->
 +
                    <div class="block-content-brief">
 +
                        <div>
 +
                            As an executor for killing the cell labeled as slackers, killer device
 +
                            should include the detecting part like in-promoter interacting
 +
                            with inhibitor protein as well as the killing part like toxin gene
 +
                            mazF and hokD.
 +
                        </div>
 +
                        <div>
 +
                            Ways of self-killing:
 +
                        </div>
 +
                        <div>
 +
                            1) toxic protein
 +
                        </div>
 +
                        <div>
 +
                            2) sgRNA targeting genome of strains with no NHEJ repair system
 +
                        </div>
 +
                        <div>
 +
                            3) sgRNA targeting essential genes
 +
                        </div>
 +
                    </div>
 +
 
 +
                    <div class="block-content-item">
 +
                        <!--Killer device小分块-->
 +
                        <div class="block-content-item-block">
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                    alt="ul_logo1" width="18" height="18">
 +
                                <span>PART DESIGN</span>
 +
                            </div>
 +
                            <div>
 +
                                At the first stage, we need to verify the function of two toxin proteins
 +
                                and make sure the bacteria can be killed after arabinose induction.
 +
                                To control the expression of killer gene, instead of J23119,
 +
                                arabinose inducible regulatory promoter/repressor unit [part number] is employed in our circuit.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig3.png"
 +
                                    alt="fig3" class="center-block" style="width: 60%;">
 +
                            </div>
 +
                            <div>
 +
                                After measuring the growth curve, we observed the obvious
 +
                                difference between the testing group and control group. [see results page for killer]
 +
                            </div>
 +
                            <div>
 +
                                To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with
 +
                                the killer circuit and replace the RFP for killer gene. [see design for threshold]
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/b/b4/T--BIT-China--Project--Design--fig4.png"
 +
                                    alt="fig4" class="center-block" style="width: 60%;">
 +
                            </div>
 +
                            <div>
 +
                                For the toxic protein,
 +
                                MazF and HokD were chosen as candidates. [see more about the killing mechanisms]
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
 
 +
                <!--Threshold device-->
 +
                <div class="block-title">Threshold device</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-brief">
 +
                        Inhibitor and in-promoter are used to measure and adjust
 +
                        the threshold. Before apply the threshold value into our
 +
                        model, we need to use RFP as a reporter
 +
                        gene since the expression of toxin protein is hard to quantify. (know more about our model)
 +
                    </div>
 +
                    <div class="content-block-item">
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                alt="ul_logo1" width="18" height="18">
 +
                            <span>Goal</span>
 +
                        </div>
 +
                        <div>
 +
                            (1) Build three parallel circuits employing different inhibitor proteins
 +
                        </div>
 +
                        <div>
 +
                            (2) Find out the initial thresholds by measuring RFP intensity
 +
                        </div>
 +
                        <div>
 +
                            (3) Provide experimental data for modeling part
 +
                        </div>
 +
                    </div>
 +
 
 +
                    <div class="content-block-item">
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                alt="ul_logo1" width="18" height="18">
 +
                            <span>Part Design</span>
 +
                        </div>
 +
                        <div>
 +
                            The gene circuits are shown in Fig.5.
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/7/7e/T--BIT-China--Project--Design--fig5.png"
 +
                                alt="fig5" class="center-block" style="width: 60%;">
 +
                        </div>
 +
                        <div>
 +
                            (1) The concentration of inducer arabinose is regarded as an input signal,
 +
                            thus Part:BBa_K808000 Arac-pBAD is used to control the expression of inhibitor proteins
 +
                        </div>
 +
                        <div>
 +
                            (2) Three combinations of inhibitor and in-promoter are selected to prove
 +
                            the concept of plasmid quorum sensing [see inhibitor mechanisms]
 +
                        </div>
 +
                        <div>
 +
                            (3) RFP is used as an indicator
 +
                        </div>
 +
                        <div>
 +
                            We assume that:
 +
                        </div>
 +
                        <div>
 +
                            The high concentration of inducers can simulate high concentration
 +
                            of plasmids due to the same effect of producing more inhibitor proteins.
 +
                        </div>
 +
                        <div>
 +
                            By adding different concentration of arabinose, we can measure the fluorescence intensity
 +
                            of RFP and find out the initial thresholds under control of certain inhibitor proteins.
 +
                        </div>
 +
                    </div>
 +
 
 +
                </div>
 +
 
 +
                <!--Upgraded P-SLACKiller-->
 +
                <div class="block-title">Upgraded P-SLACKiller</div>
 +
                <!--Upgraded P-SLACKiller 问题描述-->
 +
                <div class="problem-txt">
 +
                    <div class="problem-title">Why do we need to optimize our project?</div>
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"
 +
                            alt="ul_logo1" width="18" height="18">
 +
                    <span>It’s possible to produce generation of plasmid-free cells.
 +
                        Thus, the killer device should be integrated into the genome. [see reombination]</span>
 +
                    </div>
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"
 +
                            alt="ul_logo1" width="18" height="18">
 +
                    <span> To meet various needs with different plasmid numbers,
 +
                        we need to adjust the initial threshold
 +
                        through replacement of RBS as well as in-promoter mutation. [see mutation]</span>
 +
                    </div>
 +
                </div>
 +
                <div class="block-title">Recombination</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-brief">
 +
                    Recombineering (recombination-mediated genetic engineering) is
 +
                    an efficient molecular engineering technique used for gene
 +
                    replacement, deletion and insertion.
 +
                    </div>
 +
 
 +
                    <div class="content-block-item">
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                alt="ul_logo1" width="18" height="18">
 +
                            <span>Ways: a linear plus circular reaction</span>
 +
                        </div>
 +
                        <div>
 +
                            A：Employment of traditional λ-Red recombination
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/6/6d/T--BIT-China--Project--Design--ul_logo3.png"
 +
                                alt="ul_logo3" width="18" height="18">
 +
                            <span>PART DESIGN</span>
 +
                        </div>
 +
                        <div>
 +
                            The basic circuit of upgraded P-SLACKiller is shown in Fig.6.
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--Project--Design--fig6.png"
 +
                                alt="fig6" class="center-block" style="width: 60%;">
 +
                        </div>
 +
                        <div>
 +
                            Compared with the original one, two homologous arms (50bp) are added.
 +
                            With the help of lambda Red recombination system, the killer part can be integrated into genome,
 +
                            preventing the whole system from being invalid. The final gene circuit is shown in Fig.7.
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/c/cd/T--BIT-China--Project--Design--fig7.png"
 +
                                alt="fig7" class="center-block" style="width: 60%;">
 +
                        </div>
 +
 
 +
                        <!--B 部分-->
 +
                        <div>
 +
                            B:：CRISPR-Cas9 coupling λ-Red recombineering
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/6/6d/T--BIT-China--Project--Design--ul_logo3.png"
 +
                                alt="ul_logo3" width="18" height="18">
 +
                            <span>PART DESIGN</span>
 +
                        </div>
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/2/2a/T--BIT-China--Project--Design--fig8.png"
 +
                                alt="fig8" class="center-block" style="width: 60%;">
 +
                        </div>
 +
                        <div>
 +
                            Difference between the two methods above: Traditional way use the resistance
 +
                            gene as a selection marker, while the CRISPR coupled way
 +
                            introduce a double-strand break (DSB) into the strain with no recombination occurred.
 +
                        </div>
 +
                    </div>
 +
 
 +
                    <div class="content-block-item">
 +
                        <div>
 +
                            <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                                alt="ul_logo1" width="18" height="18">
 +
                            <span>Application:</span>
 +
                        </div>
 +
                        <div>
 +
                            In our project, we tried both the two ways to test the integration
 +
                            efficiency. However, the CRISPR coupled
 +
                            way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction.
 +
                        </div>
 +
                        <div>
 +
                            We tried the traditional way with the pKD46 plasmid
 +
                            provided by Chun Li’s lab and got the positive results. [results for recombination]
 +
                        </div>
 +
                        <div>
 +
                            Two main factors will influence the recombination efficiency: the target site on genome
 +
                            and the length of homologous arms between donor fragment. [recombination protocol]
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
                <div class="block-title">Promoter mutation</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-brief">
 +
                        For inductive promoters, two kinds of region are significant
 +
                        to its transcription strength. One is common -10 and -35 region
 +
                        which can firmly bind to RNA polymerase, the other is
 +
                        operon region binding with regulatory factors like
 +
                        inhibitor proteins. [see mutation mechanisms]
 +
                    </div>
 +
 
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/2/21/T--BIT-China--Project--Design--ul_logo2.png"
 +
                            alt="ul_logo1" width="18" height="18">
 +
                        <span> Aim:</span>
 +
                    </div>
 +
                    <div>
 +
                        (1) To adjust threshold through changing in-promoter’s strength
 +
                    </div>
 +
                    <div>
 +
                        (2) Tied to specific promoter like pTet, build a mutants library and improve the existing part by measuring the strength through RFP
 +
                    </div>
 +
 
 +
                    <div>
 +
                        <img src="https://static.igem.org/mediawiki/2016/0/08/T--BIT-China--Project--Design--fig9.png"
 +
                            alt="fig9" class="center-block" style="width: 60%;">
 +
                    </div>
 +
                    <div>
 +
                        After analyzing the promoter region of pTet, we designed
 +
                        random primers to mutate its -35 region.
 +
                        RFP intensity is measured to indicate the strength of
 +
                        mutated promoters. [see mutation results]
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
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Revision as of 10:26, 7 October 2016

backtop
Design
What is the problem?
ul_logo1 Plasmid segragational instability has been the limit step for large-scale protein production and bioremediation. Use of antibiotics for plasmid retention is not practical in this situation.
ul_logo1 High copy number (hcn) plasmids are lost from population at a high rate due to the large metabolic burden and lack of nature factors for plasmid maintenance.
Our P-SLACKiller
ul_logo1 IDEA
Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller). Use the inhibitor protein as a signal indicating plasmid numbers, when the plasmid concentration reduces to a threshold, the switch of killer gene will turn on.
fig1
ul_logo1 PART DESIGN
The basic circuit is shown in Fig.2, the genetic locus of functional gene can be substituted on the basis of production demand in the future. Inhibitor and in-promoter are key to control the threshold. When the signal of plasmid losing is detected, the executor killer will play a role.
fig2
Killer device
As an executor for killing the cell labeled as slackers, killer device should include the detecting part like in-promoter interacting with inhibitor protein as well as the killing part like toxin gene mazF and hokD.
Ways of self-killing:
1) toxic protein
2) sgRNA targeting genome of strains with no NHEJ repair system
3) sgRNA targeting essential genes
ul_logo1 PART DESIGN
At the first stage, we need to verify the function of two toxin proteins and make sure the bacteria can be killed after arabinose induction. To control the expression of killer gene, instead of J23119, arabinose inducible regulatory promoter/repressor unit [part number] is employed in our circuit.
fig3
After measuring the growth curve, we observed the obvious difference between the testing group and control group. [see results page for killer]
To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with the killer circuit and replace the RFP for killer gene. [see design for threshold]
fig4
For the toxic protein, MazF and HokD were chosen as candidates. [see more about the killing mechanisms]
Threshold device
Inhibitor and in-promoter are used to measure and adjust the threshold. Before apply the threshold value into our model, we need to use RFP as a reporter gene since the expression of toxin protein is hard to quantify. (know more about our model)
ul_logo1 Goal
(1) Build three parallel circuits employing different inhibitor proteins
(2) Find out the initial thresholds by measuring RFP intensity
(3) Provide experimental data for modeling part
ul_logo1 Part Design
The gene circuits are shown in Fig.5.
fig5
(1) The concentration of inducer arabinose is regarded as an input signal, thus Part:BBa_K808000 Arac-pBAD is used to control the expression of inhibitor proteins
(2) Three combinations of inhibitor and in-promoter are selected to prove the concept of plasmid quorum sensing [see inhibitor mechanisms]
(3) RFP is used as an indicator
We assume that:
The high concentration of inducers can simulate high concentration of plasmids due to the same effect of producing more inhibitor proteins.
By adding different concentration of arabinose, we can measure the fluorescence intensity of RFP and find out the initial thresholds under control of certain inhibitor proteins.
Upgraded P-SLACKiller
Why do we need to optimize our project?
ul_logo1 It’s possible to produce generation of plasmid-free cells. Thus, the killer device should be integrated into the genome. [see reombination]
ul_logo1 To meet various needs with different plasmid numbers, we need to adjust the initial threshold through replacement of RBS as well as in-promoter mutation. [see mutation]
Recombination
Recombineering (recombination-mediated genetic engineering) is an efficient molecular engineering technique used for gene replacement, deletion and insertion.
ul_logo1 Ways: a linear plus circular reaction
A：Employment of traditional λ-Red recombination
ul_logo3 PART DESIGN
The basic circuit of upgraded P-SLACKiller is shown in Fig.6.
fig6
Compared with the original one, two homologous arms (50bp) are added. With the help of lambda Red recombination system, the killer part can be integrated into genome, preventing the whole system from being invalid. The final gene circuit is shown in Fig.7.
fig7
B:：CRISPR-Cas9 coupling λ-Red recombineering
ul_logo3 PART DESIGN
fig8
Difference between the two methods above: Traditional way use the resistance gene as a selection marker, while the CRISPR coupled way introduce a double-strand break (DSB) into the strain with no recombination occurred.
ul_logo1 Application:
In our project, we tried both the two ways to test the integration efficiency. However, the CRISPR coupled way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction.
We tried the traditional way with the pKD46 plasmid provided by Chun Li’s lab and got the positive results. [results for recombination]
Two main factors will influence the recombination efficiency: the target site on genome and the length of homologous arms between donor fragment. [recombination protocol]
Promoter mutation
For inductive promoters, two kinds of region are significant to its transcription strength. One is common -10 and -35 region which can firmly bind to RNA polymerase, the other is operon region binding with regulatory factors like inhibitor proteins. [see mutation mechanisms]
ul_logo1 Aim:
(1) To adjust threshold through changing in-promoter’s strength
(2) Tied to specific promoter like pTet, build a mutants library and improve the existing part by measuring the strength through RFP
fig9
After analyzing the promoter region of pTet, we designed random primers to mutate its -35 region. RFP intensity is measured to indicate the strength of mutated promoters. [see mutation results]

P-SLACKiller

· Work or Die
· Monitor Plasmid Numbers
· Kill the Slacker
· Maintain the Better Biosystem

Useful Links

Sponsors

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Get in touch

Email: iGEM_BIT_China@163.com
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