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| - | <h2>Overview</h2> | + | <h2>Why we want to improve it?</h2> |
| | + | <p><center><strong>Whether the Freiburg's design is efficient or not</strong></center></p> |
| | + | <p>According to the experimental record of Freiburg, the success rate is higher than 95%(32/33). </p><p>However, this result apparently lacks statistical significance.</p> |
| | + | <p>In the result section, they emphasize that there is a light band at 1200bp, which they believe could indicate that the Golden Gate connection works well. However, after conducting several experiments by ourselves, we find that the key point to indicate whether Golden Gate connection works is not the band at 1200bp. If the band is not clear and specific in the gel, it indicates the experiment doesn’t go well. We can easily find several light bands under the band of 1200bp. Moreover, the second light band is somewhat lighter than the band at 1200bp. Although the Freiburg can explain the results with the repeatability of the TALE sequence, we suppose that the possibility of the mismatch of the sticky ends still can’t be excluded. Frankly speaking, we try to believe that they really made it, but if the success cannot be repeated, there must be something wrong with their system.</p> |
| | + | <p><center><strong>The protocol we take to connect the parts of TALE</strong></center></p> |
| | + | <ol><li> Freiburg's protocol</li> |
| | + | <li> Restriction enzyme digestion of plasmid and TAL repeats and gel extraction respectively. By the mole ratio, plasmid to TALE is 1 to 5 and TALE to TALE is 1 to 1. Ligation with T4 ligase in 22 ℃over night.</li> |
| | + | <li>The same ratio of plasmid and TALE repeat, but add the TALE repeats one by one and ligation in 22 ℃, 30 minutes</li> |
| | + | <li>Every two parts connect at one time, and try to make three intermediates of 400bp, and then mix the plasmid to make the complete TALE.</li> |
| | + | <li>The same ratio and ligation with the program of 22℃ 2min, 40℃ 30,25 repeats.</li></ol> |
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| - | <p>Most biochemical reactions rely on the enzyme. An enzyme system composed of various enzymes is needed to participate in the complex metabolic pathways. As a result, they tend to form a complex, significantly increasing the reaction efficiency by increasing enzyme-substrate contact. However, in applicational fields such as industry and environment, enzymes we directly use can not form a multi-enzyme complex on most occasions, so the reaction efficiency is not satisfactory. </p>
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| - | <p>This year, our project intends to achieve protein polymerization with the help of TAL effector, a DNA-binding protein, which can recognize specific nucleic acid sequences. With circular DNA, plasmid, as the connection medium, expressed proteins in the host can form a complex selectively, so that a variety of enzyme combinations can be used to complete different tasks. Compared to traditional methods, the advantages of the project lie in selective polymerization of proteins, and the ability to mediate polymerization between more proteins.</p>
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| - | <p>Enzyme polymerization: In prokaryotic cells, enzymes are freely dispersed. However, our fused protein can be effectively anchored and brought closer to each other. In this way, it can shorten the time for substrates to transport between each enzyme in the pathway, reduce the energy loss of substrate mobilization, and enhance the reaction efficiency.</p>
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| - | <p>Enzyme maximization: Because TAL’s recognition sites are fairly abundant and of little specificity, we will be more likely to find corresponding recognition sites on a plasmid. Since there might be more qualified sequences on a plasmid, one of our goals is to find as many of them as possible, so that more enzymes can be polymerized on the same plasmid. This provides us with multiple ways to utilize the element.</p>
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| - | <p>Enzyme polymerization plus maximization: As mentioned earlier, our project can achieve enzyme polymerization as well as maximization. Hence, we attempt to combine these two highlights in order to deliver greater value. Among all the enzymes involved in a metabolic pathway, we can factitiously combine several (three, four, five or even more) specific enzymes in order to optimize their reaction synergy.</p>
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| - | <p>Based on the above-mentioned facts, we can draw the conclusion that our project has various flexible and extensive applicational prospects, due to its fundamentality. Through preliminary consideration, we believe our project can be applied to various aspects, including food processing, biofuel production, water treatment and bio-pharmaceuticals.</p>
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| - | <h2 id="schematicintroduction">Schematic introduction</h2>
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| - | <p>As mentioned in the overview, in order for the protein to bind to the plasmid–the connector, we have designed two sorts of delicat fusion proteins–the connectee. The first kind of connectee is free while the second is anchored to the cell membrane. Both of them consist of various sections, which are shown in the schematic diagrams below.<br/>
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| - | !!(插图2幅：融合蛋白的结构，可参考海报或ppt。第一幅图只包括Enzyme-FL-TAL，第二幅图包括所有的，ssDsbA-FP-HL-Lgt-FL-Enzyme-FL-TAL)</p>
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| - | <ol>
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| - | <li>TAL protein, the transactivator-like protein. According to its special structure, the TAL protein plays a great role in DNA binding. The 2012 Freiburg iGEM team has offered us a whole set of 96 TAL-protein direpeat bioparts, with which we are supposed to build functional TAL proteins. Since each TAL protein can identify a 14-nucleotide target sequence, the first and fourteenth nucleotide being Thymin, all the 96 parts can be used to identify more than 16 million different nucleotide sequences, which makes it very convenient for us to choose a sequence for the fusion protein to bind to.</li>
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| - | <li>The enzyme we want to bring together. Different proteins that bind to the same plasmid contain different enzymes from the same metabolic pathway.<br/>
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| - | With these two parts combined by a flexible linker, we can obtain the first type of connectee, that is free from the cell membrane. But if we want to bind the fusion protein to it, at least two more sections are needed, which are listed below.</li>
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| - | <li>SsDsbA, a signal peptide at the N-terminal of the protein. It can direct the fusion protein to the preiplasm.</li>
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| - | <li>Lgt, a transmembrane protein whose function has been identified in previous iGEM projects. All these four sections, together with linkers, enable us to build the second type of fusion protein, which can be anchored to the cell membrane. Besides, FP, the fluorescent protein is also needed when we want to detect whether the fusion protein is expressed. During detection, FP is expressed between ssDsbA and Lgt while enzyme is not linked to the whole fusion protein in order to ensure its proper size. For different connectee, we can use different fluorescent protein to detect the expression successively, so that they can be distinguished easily.<br/>
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| - | In conclusion, with the help of our artificial multi-enzyme complex systems, we are sure to improve the dynamic characteristics of metabolic reactions in many applicational fields.</li>
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| - | </ol>
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| | <h2 id="reference">Reference</h2> | | <h2 id="reference">Reference</h2> |
Why we want to improve it?
Whether the Freiburg's design is efficient or not
According to the experimental record of Freiburg, the success rate is higher than 95%(32/33).
However, this result apparently lacks statistical significance.
In the result section, they emphasize that there is a light band at 1200bp, which they believe could indicate that the Golden Gate connection works well. However, after conducting several experiments by ourselves, we find that the key point to indicate whether Golden Gate connection works is not the band at 1200bp. If the band is not clear and specific in the gel, it indicates the experiment doesn’t go well. We can easily find several light bands under the band of 1200bp. Moreover, the second light band is somewhat lighter than the band at 1200bp. Although the Freiburg can explain the results with the repeatability of the TALE sequence, we suppose that the possibility of the mismatch of the sticky ends still can’t be excluded. Frankly speaking, we try to believe that they really made it, but if the success cannot be repeated, there must be something wrong with their system.
The protocol we take to connect the parts of TALE
- Freiburg's protocol
- Restriction enzyme digestion of plasmid and TAL repeats and gel extraction respectively. By the mole ratio, plasmid to TALE is 1 to 5 and TALE to TALE is 1 to 1. Ligation with T4 ligase in 22 ℃over night.
- The same ratio of plasmid and TALE repeat, but add the TALE repeats one by one and ligation in 22 ℃, 30 minutes
- Every two parts connect at one time, and try to make three intermediates of 400bp, and then mix the plasmid to make the complete TALE.
- The same ratio and ligation with the program of 22℃ 2min, 40℃ 30,25 repeats.
Reference
- GONTERO, Brigitte, María Luz CÁRDENAS, and Jacques RICARD. “A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle.” European journal of biochemistry 173.2 (1988): 437–443.
- Bogdanove, Adam J., and Daniel F. Voytas. “TAL effectors: customizable proteins for DNA targeting.” Science 333.6051 (2011): 1843–1846.
- !!参考往届iGEM的wiki不知道是否需要写上去，以及应该用什么样的格式
- Pailler, Jérémy, et al. “Phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.” Journal of bacteriology 194.9 (2012): 2142–2151.
- Deng, Dong, et al. “Structural basis for sequence-specific recognition of DNA by TAL effectors.” Science 335.6069 (2012): 720–723.
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