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| <div class="content"> | | <div class="content"> |
| + | <div class="jiao" > |
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| + | <div class="projtile_only"> |
| + | <h2 id="subtitleyjn1">TAL Improvement</h2></br> |
| + | <h2 id="subtitleyjn2">——More Effective Golden Gate Cloning </h2> |
| + | <center><p>This year our team not only focused on our project and bioparts but also improved several existing parts designed by iGEM12_Freiburg. When we used their parts and followed their protocols, we found that it was hard for us to repeat their success. There were a lot of difficulties on our way to constructing a TALE. After discussion and thinking, our team decided to improve the existing TAL parts by finding out new and better sticky ends. Moreover, our method can be used in border ways when people want to connect some components by Golden Gate method.</p></center> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dianweidian3" title="Why we want to improve it?"> |
| + | <center><h2>Why improve it?</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dianweidian4" title="How do we connect certain monomers?"> |
| + | <center> <h2>How to connect monomer?</h2></center></a> |
| + | </div> |
| + | |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dianweidian5" title="Why not other sticky ends?"> |
| + | <center><h2>Why not other sticky ends?</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dianweidian6" title="How to improve the Golden Gate sticky ends? A big Table!"> |
| + | <center><h2 style="LINE-HEIGHT:30px; margin-left:7%; margin-right:7% ;" >How to improve the Golden Gate sticky ends?</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dianweidian7" title="Best choice for seven sticky ends on TALE protein"> |
| + | <center><h2 style="LINE-HEIGHT:30px; margin-left:7%; margin-right:7% ;">Best choice for seven sticky ends on TALE protein</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile" id="dianweidian3" > |
| + | <a href="#dianweidian8" title="Reconstruct DNA Sequence"> |
| + | <center><h2>Reconstruct DNA sequences</h2></br></center></a> |
| + | </div> |
| + | |
| + | |
| + | </div> |
| + | |
| + | <div style="clear:both;"></div> |
| + | |
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| <article class="post__article"> | | <article class="post__article"> |
- | <h2>Why we want to improve it?</h2> | + | |
- | <p><center><strong><span style="font-size:22px;color:blue">Whether the Freiburg's design is efficient or not</span></strong></center></p> | + | <h2 >Why we want to improve it?</h2> |
- | <p>According to the experimental record of Freiburg, the success rate is higher than 95%(32/33). However, this result, to some degree, lacks statistical significance.</p> | + | <h3>Whether the Freiburg's design is efficient or not</h3> |
- | <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. You can view <a href="https://2012.igem.org/Team:Freiburg/Project/Experiments">Detail information</a> in iGEM2012 Freiburg wiki.</p> | + | <p>According to the experimental record of Freiburg, the success rate is higher than 95%(32/33). However, this result, to some degree, lacks statistical significance.</p> |
- | <center><img src="https://static.igem.org/mediawiki/2014/5/57/Freiburg_result_picture.png"></img></center> | + | <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 found 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. You can view <a href="https://2012.igem.org/Team:Freiburg/Project/Experiments">Detail information</a> in iGEM2012 Freiburg wiki.</p> |
- | <p><center><strong>The protocol we take to connect the parts of TALE</strong></center></p> | + | <center><img alt="Freiburg gel result" src="https://static.igem.org/mediawiki/2014/5/57/Freiburg_result_picture.png"width=400px vspace=20px></img></center> |
- | <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> | + | <center><small><strong> Figure 1.4.1 2012 Freiburg gel result</strong></small></center> |
- | <li>The same ratio of plasmid and TALE repeat, but add the TALE repeats one by one and ligation in 22 ℃, 30 minutes</li> | + | <h3>The protocols we took to connect the parts of TALE</h3> |
- | <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> | + | <p>1. Freiburg's protocol</p> |
- | <p><center><strong>The motivation to debug 2012 Freiburg’s parts</strong></center></p> | + | <p>2. Restriction enzyme digestion of plasmid and TAL direpeats, followed by gel extraction. 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.</p> |
- | <p>Unfortunately, all of our attempts failed. We didn’t manage to make a complete TALE, or even make two of them together. However, what is important for us is that when we try the 5th protocol, we notice an unexpected result. When we analyze the sequence result, we find that our left adaptor, 1st part and right adaptor connect together. Why do we get this result? We notice that their sticky end is TGAC, GCTC, and ACTC. That is to say, GCTC and ACTC connect with each other by mistake. In another word, if the sticky ends are very similar, they probably connect with each other. Although we failed again, the result gives us confidence to debug 2012 Freiburg's parts. </p> | + | <p>3. With the same ratio of plasmid and TALE direpeats, add the TALE direpeats one by one and connect them in 22 ℃, 30 minutes.</p> |
- | <h2>How do we connect certain monomer? </h2> | + | <p>4. Every two parts connect at one time, and try to make three intermediates of 400bp. And then mix the plasmids to make the complete TALE.</p> |
- | <p><center><strong>Some advanced tips for TALE protein</strong></center><p> | + | <p>5. The same ratio and connect following the program of 22℃ 2min, 40℃ 30s, 25 repeats.</p> |
- | <ol>
| + | |
- | <li>Given a sample sequence with repeating amino acids: | + | <h3>The motivation to improve 2012 Freiburg’s parts</h3> |
- | <br/> | + | |
- | <img src="https://static.igem.org/mediawiki/2014/f/f2/SJTU14_tal_improvment_1.png"></img> | + | <p id="dianweidian4">Unfortunately, all of our attempts failed. We didn’t manage to make a complete TALE, or even make two of them together. However, what is important for us is that when we tried the 5th protocol, we noticed an unexpected result. When we analyzed the sequence result, we found that our left adaptor, 1st part and right adaptor connected together. Why did we get this result? We noticed that their sticky ends are TGAC, GCTC, and ACTC. That is to say, GCTC and ACTC might have connected with each other by mistake. In another word, if the sticky ends are very similar, they probably connect with each other. Although we failed again, the result gave us confidence to improve 2012 Freiburg's parts. </p> |
- | <p>What XX means is that it determine the certain kind of base. For one unit of repetition, other amino acids can be identical.</p></li> | + | |
- | <li>A fully functional TALE protein contains one sequence, that does not have repetitive units, recognizing base T, and similar sequence but is only half length as its end. That is, one complete TALE protein is able to recognize certain number of repetitive units and two bases.</li> | + | <h2>How do we connect certain monomer? </h2> |
- | <li>The length that can be recognized is not strictly twelve or fourteen. According to the published results, the length and certain sequence are dependent on number and type of monomer.</li> | + | <h3>Some advanced tips for TALE protein</h3> |
- | </ol> | + | |
- | <p>We can gather 96 bioparts based on Freiburg, and each part has its counterproductive base on certain location(1,2,3,4,5 or 6). By picking two bases on certain location, we are able to design one TALE protein sequence.</p> | + | <p>1. Given a sample sequence with repeating amino acids:</p> |
- | <br/>
| + | <center><img src="https://static.igem.org/mediawiki/2014/f/f2/SJTU14_tal_improvment_1.png"></img></center> |
- | <p><center><strong> Previous Review: Freiburg’s way of connection</strong></center><p> | + | |
- | <p>The main principles of connection is built upon the idea of Golden Gate Connection.( Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).)</p> | + | <center><small><strong> Figure 1.4.2 TALE amino acid sequence</strong></small></center> |
- | <p>The gust of these procedures is more related to one type of restriction enzyme, type II Restriction Enzyme, especially BsmBI enzyme.</p> | + | <p>What XX means is that it determines the certain kind of base. For one unit of repetition, other amino acids can be identical.</p> |
- | <center><img src="https://static.igem.org/mediawiki/2014/5/5b/SJTU14_tal_improvment_2.png"></img></center> | + | <p>2. A fully functional TALE protein contains a segment of sequence before repetitive units, recognizing first base T. It also contains a similar segment sequence but it is only half length as the repetitive unit which can recognize the last base T. </p> |
- | <p>The main feature of this enzyme is the recognition sequence is on only one side of cleavage site. It provides the way which can be used to get certain incision without damaging the whole sequence. The sticky end has 4bp base, and it could be designed even for combination of multiple sticky end. That feature is fancy at first, but we cannot regardless its latent shortcomings. </p> | + | <p>3. The length that can be recognized is not strictly twelve or fourteen. According to the published results, the length of recognition sequence are dependent on the number of monomer.</p><br> |
- | <p>Let’s analyze the example (AA1) provided by Freiburg. </p> | + | |
- | <center><img src="https://static.igem.org/mediawiki/2014/b/bc/SJTU14_tal_improvment_3.png | + | <p>We can gather 96 bioparts based on Freiburg, and each part has its counterproductive sticky ends base on certain location(1,2,3,4,5 or 6). By picking every two bases on certain location, we are able to design one TALE protein sequence.</p> |
- | "></img><center>
| + | |
- | <p><small>The underlined parts are recognized by BsmBI. Vertical bar(|) is the cutting position. As for this sample, TGAC is one sticky end which can combine with other seven sticky ends.</small></p> | + | <h3>Previous Review: Freiburg’s way of connection</h3> |
- | <p><center><strong> Evaluate seven sticky ends designed by 2012 Freiburg</strong></center><p> | + | |
- | <p>2012 Freiburg's parts have seven sticky ends:</p> | + | <p>The main principle of connection is built upon the idea of Golden Gate Connection.( Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).)</p> |
- | <p><center>TGAC,GCTC,CTTG,GCTT,ACTG,CCTG,ACTC<center></p> | + | <p>The key point of their procedures is a type II Restriction Enzyme, BsmBI enzyme.</p> |
- | <p>We all know that certain two parts can combine together, under base-pair rule. However, whether it is possible that unpaired sticky ends can bind together? In fact, the more similar they are, the more possibility that can form new but error base pairs. | + | <center><img src="https://static.igem.org/mediawiki/2014/5/5b/SJTU14_tal_improvment_2.png" ></img></center> |
- | Spired by BLAST algorithm, we calculate the similarity of each other sticky ends. </p>
| + | |
- | </article> | + | <center><small><strong> Figure 1.4.3 BsmBI recognition site</strong></small></center> |
| + | <p>The main feature of this enzyme is that the recognition sequence is on only one side of cleavage site. It provides the way which can be used to get certain sticky ends with out breaking the whole sequence. The sticky end has 4bp, and it could be designed for combination of multiple sticky ends. That feature is excellent at first, but we cannot ignore its latent shortcomings. </p> |
| + | <p>Let’s analyze the example (AA1) provided by Freiburg. </p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/b/bc/SJTU14_tal_improvment_3.png" width=700px></img></center> |
| + | |
| + | <center><small><strong> Figure 1.4.4 DNA sequence of AA1 part</strong></small></center> |
| + | <p><small><i>The underlined sequence is recognized by BsmBI. Vertical bar(|) is the cutting position. As for this sample, TGAC is one sticky end which can combine with six other sticky ends.</i></small></p> |
| + | <h3>Evaluate seven sticky ends designed by 2012 Freiburg</h3> |
| + | <p>2012 Freiburg's parts have seven sticky ends:</p> |
| + | <p><center>TGAC,GCTC,CTTG,GCTT,ACTG,CCTG,ACTC</center></p> |
| + | <p>We all know that certain two parts can combine together, under the principle of complementary base pairing. However, is it possible that not totally matched sticky ends can bind together? We found, in fact, the more similar they are, the more possibility that they can form new but incorrect base pairs. |
| + | Inspired by BLAST algorithm, we evaluated the similarity of every pair of sticky ends. </p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/8/8e/TAL%E7%B2%98%E6%80%A7%E6%9C%AB%E7%AB%AF%E8%A1%A8%E6%A0%BC.png" width=400px></img></center> |
| + | <center><small><strong> Figure 1.4.5 Strict rules score table </strong></small></center> |
| + | <p id="dianweidian5">The higher score, the higher similarity, and the higher possibility of mismatch. |
| + | The table shows that more than 30% of pairs’ score is equal to 3, which means that the possibility of mismatch cannot be neglected. |
| + | |
| + | Even if we employ the relatively loose rule to evaluate the similarity, we can still find that error rates cannot be neglected.</p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/9/9b/Tal_%E8%A1%A8%E6%A0%BC%E7%B2%98%E6%80%A7%E6%9C%AB%E7%AB%AF2.png" width=400px></img></center> |
| + | <center><small><strong>Figure 1.4.6 Loose rules score table </strong></small></center> |
| + | <h2> Why not other sticky ends?</h2> |
| + | <h3>The reason why Freiburg used these sticky ends</h3> |
| + | <p>Failed to contact the original designers of these sticky ends, what we can do is just to find feasible advantages of these combinations.</p> |
| + | <p>Let's look at the TALE direpeat unit amino acids sequence:</p> |
| + | <p><center>LTPEQVVAIAS(XX)GGKQALETVQRLLPVLCQAHG(34aa)</center></p> |
| + | <p id="dianweidian6">The first amino acid is Leu, which is essential for all connection process. There are six different codons for Leu. </p> |
| + | <p><center>UUA,UUG,CUU,CUC,CUA,CUG</center></p> |
| + | |
| + | <p>The 2012 Freiburg project's sticky ends:</p> |
| + | <p><center>(C)TGAC,GCTC,CTTG,GCTT,ACTG,CCTG,ACTC</center></p> |
| + | <p>The feature of Degeneracy has helped to design seven sticky ends. However, since the codons for identical amino acid are highly similar, |
| + | this feature, for experimental scientists, is a double-edged sword.</p> |
| + | <h2>How to improve the Golden Gate sticky ends? A big Table!</h2> |
| + | |
| + | <p>Three key questions need to be answered:</p> |
| + | <p>1. Is it possible to find perfect match pair?</p> |
| + | <p>2. Can we find a certain number of sticky ends with least mismatch possibility?</p> |
| + | <p>3. How to make this sticky-end score table?</p> |
| + | |
| + | <h3>Key algorithms derived from BLAST algorithm</h3> |
| + | <p>Loose rule: Match: 1; Mismatch: 0; Gap: 0</p> |
| + | <p>Strict rule: Match: 1; Mismatch: 0; Gap: 1</p> |
| + | <p>The sticky end is composed of four bases, which means that we can design 256 types of sticky ends, which are represented as a 256*256 table. </p> |
| + | <h3>Find target groups of sticky ends</h3> |
| + | <p>To solve the TALE parts problem, we need find seven sticky ends, and the similarity score of each pair in them are less than or equal to 1.</p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/1/12/Choices_for_group.png" width=400px></img></center> |
| + | <center><small><strong>Figure 1.4.7 Sticky ends choices table </strong></small></center> |
| + | <p>When we select Strict Algorithm to find these ends, it is impossible to find seven sticky ends, that each pair of them has score no more than 1. So we have to select Loose Algorithm.</p> |
| + | <h3 > Convert four-basepair sticky ends to amino acid pairs</h3> |
| + | <p>We care about whether two amino acids located on our target sequence, rather than the 4bp. So we should convert the sticky ends sequence to amino acid pairs.</p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/4/45/Amino_acid_table.png"width=600px id="dianweidian7"></img></center> |
| + | |
| + | <center><small><strong>Figure 1.4.8 4bp sticky ends convert to Amino acid table </strong></small></center> |
| + | <p>Based on the above table, we are able to calculate the total scores of each combination and find the best one.</p> |
| + | <h2>Best choice for seven sticky ends on TALE protein</h2> |
| + | <p>Best combination: |
| + | <center>AAAA, AGGG, GTAC, GCTC, TTTT, TCGA, CCCC</center></p> |
| + | <p>Scores Table(Loose rule):</p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/e/e0/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7_2014-10-16_%E4%B8%8B%E5%8D%883.24.40.png" width=400px></img></center> |
| + | |
| + | <center><small><strong>Figure 1.4.9 Best combination score table </strong></small></center> |
| + | |
| + | <p>Position in TALE amino acids sequence:</p> |
| + | <center><img src="https://static.igem.org/mediawiki/2014/e/e1/Sticky3333.png" width="500px" id="dianweidian8"></img></center> |
| + | |
| + | |
| + | <center><small><strong>Figure 1.4.10 Position in TALE amino acids sequence </strong></small></center> |
| + | |
| + | <h2>Reconstruct DNA sequences</h2> |
| + | |
| + | <p>Two main factors to reconstruct DNA sequences:<br> |
| + | 1.Use the table of best combination and rearrange the sticky ends according to your demand.<br> |
| + | 2.There are no BsmBI recognition sequence in the reconstruct DNA sequence.<br> |
| + | Final DNA Sequence for NEW TALE protein:</p> |
| + | <pre> |
| + | 1 CTGACCCCGG AACAGGTGGT GGCCATTGCA AGCAACGGTG GTGGCAAGCA GGCCCTGGAG |
| + | 61 ACAGTCCAAC GGCTGCTTCC GGTTCTGTGT CAGGCCCACG GCCTGACTCC AGAACAAGTG |
| + | 121 GTTGCTATCG CCAGCCACGA TGGCGGAAAA CAAGCCCTCG AAACCGTGCA GCGCCTGCTT |
| + | 181 CCGGTGCTGT GTCAGGCCCA CGGGCTCACC CCGGAACAGG TGGTGGCCAT CGCATCTAAC |
| + | 241 AATGGCGGTA AGCAGGCACT GGAAACAGTG CAGCGCCTGC TTCCGGTCCT GTGTCAGGCT |
| + | 301 CATGGCCTGA CCCCAGAGCA GGTCGTGGCA ATTGCCTCCA ACATTGGAGG GAAGCAGGCA |
| + | 361 CTGGAGACCG TGCAGCGGCT GCTGCCGGTG CTGTGTCAGG CCCACGGCTT GACCCCGGAA |
| + | 421 CAGGTGGTGG CCATCGCCTC CAACGGCGGT GGCAAACAGG CGCTGGAAAC AGTTCAACGC |
| + | 481 CTCCTTCCGG TCCTGTGCCA GGCCCATGGT CTGACTCCAG AGCAGGTTGT GGCAATTGCA |
| + | 541 AGCAACATTG GTGGTAAACA AGCTTTGGAA ACCGTCCAGC GCTTGCTGCC AGTACTGTGT |
| + | 601 CAGGCCCACG GGCTTACCCC GGAACAGGTG GTGGCCATTG CAAGCAACGG TGGTGGCAAG |
| + | 661 CAGGCCCTGG AGACAGTCCA ACGGCTGCTT CCGGTTCTGT GTCAGGCCCA CGGCCTGACT |
| + | 721 CCAGAACAAG TGGTTGCTAT CGCCAGCCAC GATGGCGGTA AACAAGCCCT CGAAACCGTG |
| + | 781 CAGCGCCTGC TTCCGGTGCT CTGTCAGGCC CACGGACTGA CCCCGGAACA GGTGGTGGCC |
| + | 841 ATCGCCTCCA ACATTGGTGG TAAGCAAGCC CTCGAAACTG TGCAGCGGCT GCTTCCAGTC |
| + | 901 TTGTGCCAGG CTCACGGCCT GACACCGGAG CAGGTGGTTG CAATCGCGTC TAATATCGGC |
| + | 961 GGCAAACAGG CACTCGAGAC CGTGCAGCGC TTGCTTCCAG TGCTGTGTCA GGCCCACGGC |
| + | 1021 CTGACCCCGG AACAGGTGGT GGCCATCGCC TCTAACAATG GCGGCAAACA GGCATTGGAA |
| + | 1081 ACAGTTCAGC GCCTGCTGCC GGTGTTGTGT CAGGCTCACG GCCTGACTCC GGAGCAGGTT |
| + | 1141 GTGGCCATCG CAAGCCATGA TGGCGGTAAA CAAGCTCTGG AGACAGTGCA ACGCCTCTTG |
| + | 1201 CCAGTTTTGT GTCAGGCCCA CGGA |
| + | </pre> |
| + | <p>Final Amino acids remain the same:</p> |
| + | <pre> |
| + | 1 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG |
| + | 35 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG |
| + | 69 LTPEQVVAIA SNNGGKQALE TVQRLLPVLC QAHG |
| + | 103 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG |
| + | 137 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG |
| + | 171 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG |
| + | 205 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG |
| + | 239 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG |
| + | 273 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG |
| + | 307 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG |
| + | 341 LTPEQVVAIA SNNGGKQALE TVQRLLPVLC QAHG |
| + | 375 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG |
| + | </pre> |
| + | <p>Corresponding part:</p> |
| + | |
| + | <p > |
| + | <b>PART-left:</b><br> |
| + | …CTGACCCCGGAGACG |
| + | </p> |
| + | <p> |
| + | <b>PART1(150bp):</b><br> |
| + | CGTCTCGCCCCGGAACAGGTGGTGGCCATTGCAAGCAACGGTGGTGGCAAGCAGG |
| + | CCCTGGAGACAGTCCAACGGCTGCTTCCGGTTCTGTGTCAGGCCCACGGCCTGACT |
| + | CCAGAACAAGTGGTTGCTATCGTGGCGGAAAATGAGACG</p> |
| + | <p> |
| + | <b>PART2(219bp):</b><br> |
| + | CGTCTCTAAAACAAGCCCTCGAAACCGTGCAGCGCCTGCTTCCGGTGCTGTGTCAG |
| + | GCCCACGGGCTCACCCCGGAACAGGTGGTGGCCATCGCATCTAACAATGGCGGTA |
| + | AGCAGGCACTGGAAACAGTGCAGCGCCTGCTTCCGGTCCTGTGTCAGGCTCATGG |
| + | CCTGACCCCAGAGCAGGTCGTGGCAATTGCCTCCAACATTGGAGGGCGAGACG</p> |
| + | <p> |
| + | <b>PART3(262bp):</b><br> |
| + | CGTCTCTAGGGAAGCAGGCACTGGAGACCGTGCAGCGGCTGCTGCCGGTGCTGTG |
| + | TCAGGCCCACGGCTTGACCCCGGAACAGGTGGTGGCCATCGCCTCCAACGGCGGT |
| + | GGCAAACAGGCGCTGGAAACAGTTCAACGCCTCCTTCCGGTCCTGTGCCAGGCCC |
| + | ATGGTCTGACTCCAGAGCAGGTTGTGGCAATTGCAAGCAACATTGGTGGTAAACA |
| + | AGCTTTGGAAACCGTCCAGCGCTTGCTGCCAGTACGGAGACG</p></center> |
| + | <p> |
| + | |
| + | <b>PART4(224bp):</b><br> |
| + | CGTCTCCGTACTGTGTCAGGCCCACGGGCTTACCCCGGAACAGGTGGTGGCCATT |
| + | GCAAGCAACGGTGGTGGCAAGCAGGCCCTGGAGACAGTCCAACGGCTGCTTCCGG |
| + | TTCTGTGTCAGGCCCACGGCCTGACTCCAGAACAAGTGGTTGCTATCGCCAGCCA |
| + | CGATGGCGGTAAACAAGCCCTCGAAACCGTGCAGCGCCTGCTTCCGGTGCTGGGA<br> |
| + | GACG |
| + | </p> |
| + | <p> |
| + | <b>PART5(194bp):</b><br> |
| + | CGTCTCCGCTGTGTCAGGCCCACGGACTGACCCCGGAACAGGTGGTGGCCATCGC |
| + | CTCCAACATTGGTGGTAAGCAAGCCCTCGAAACTGTGCAGCGGCTGCTTCCAGTC |
| + | TTGTGCCAGGCTCACGGCCTGACACCGGAGCAGGTGGTTGCAATCGCGTCTAATA<br> |
| + | TCGGCGGCAAACAGGCACTCGATGAGACG |
| + | </p> |
| + | <p> |
| + | <b>PART6(249bp):</b><br> |
| + | CGTCTCATCGAGACCGTGCAGCGCTTGCTTCCAGTGCTGTGTCAGGCCCACGGCC |
| + | TGACCCCGGAACAGGTGGTGGCCATCGCCTCTAACAATGGCGGCAAACAGGCATT |
| + | GGAAACAGTTCAGCGCCTGCTGCCGGTGTTGTGTCAGGCTCACGGCCTGACTCCG |
| + | GAGCAGGTTGTGGCCATCGCAAGCCATGATGGCGGTAAACAAGCTCTGGAGACAG<br> |
| + | TGCAACGCCTCTTGCCAGTTTTAGAGACG</p> |
| + | <p> |
| + | |
| + | <b>PART-right:</b><br> |
| + | CGTCTCATTTTGTGTCAGGCCCACGGA...</p><br> |
| + | |
| + | |
| + | <p> |
| + | The recognition sequence of the TALE protein: |
| + | <center><font size="5" color="red">TTCGATATCAAGCT</font></center></p> |
| + | <p>All parts are under artificial synthesis process, so there is few results to present, which can prove our changes are effective. However, with the principle of complementary base pairing, our choice should be better than original version. And if you want our data or use our method to create your own best sticky ends, just contact us!</p> |
| + | |
| + | |
| + | |
| + | |
| + | |
| + | |
| + | |
| + | </article> |
| </div></html> | | </div></html> |
| + | |
| + | |
| {{Team:SJTU-BioX-Shanghai/footer}} | | {{Team:SJTU-BioX-Shanghai/footer}} |
TAL Improvement
——More Effective Golden Gate Cloning
This year our team not only focused on our project and bioparts but also improved several existing parts designed by iGEM12_Freiburg. When we used their parts and followed their protocols, we found that it was hard for us to repeat their success. There were a lot of difficulties on our way to constructing a TALE. After discussion and thinking, our team decided to improve the existing TAL parts by finding out new and better sticky ends. Moreover, our method can be used in border ways when people want to connect some components by Golden Gate method.
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, to some degree, 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 found 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. You can view Detail information in iGEM2012 Freiburg wiki.
Figure 1.4.1 2012 Freiburg gel result
The protocols we took to connect the parts of TALE
1. Freiburg's protocol
2. Restriction enzyme digestion of plasmid and TAL direpeats, followed by gel extraction. 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.
3. With the same ratio of plasmid and TALE direpeats, add the TALE direpeats one by one and connect them in 22 ℃, 30 minutes.
4. Every two parts connect at one time, and try to make three intermediates of 400bp. And then mix the plasmids to make the complete TALE.
5. The same ratio and connect following the program of 22℃ 2min, 40℃ 30s, 25 repeats.
The motivation to improve 2012 Freiburg’s parts
Unfortunately, all of our attempts failed. We didn’t manage to make a complete TALE, or even make two of them together. However, what is important for us is that when we tried the 5th protocol, we noticed an unexpected result. When we analyzed the sequence result, we found that our left adaptor, 1st part and right adaptor connected together. Why did we get this result? We noticed that their sticky ends are TGAC, GCTC, and ACTC. That is to say, GCTC and ACTC might have connected with each other by mistake. In another word, if the sticky ends are very similar, they probably connect with each other. Although we failed again, the result gave us confidence to improve 2012 Freiburg's parts.
How do we connect certain monomer?
Some advanced tips for TALE protein
1. Given a sample sequence with repeating amino acids:
Figure 1.4.2 TALE amino acid sequence
What XX means is that it determines the certain kind of base. For one unit of repetition, other amino acids can be identical.
2. A fully functional TALE protein contains a segment of sequence before repetitive units, recognizing first base T. It also contains a similar segment sequence but it is only half length as the repetitive unit which can recognize the last base T.
3. The length that can be recognized is not strictly twelve or fourteen. According to the published results, the length of recognition sequence are dependent on the number of monomer.
We can gather 96 bioparts based on Freiburg, and each part has its counterproductive sticky ends base on certain location(1,2,3,4,5 or 6). By picking every two bases on certain location, we are able to design one TALE protein sequence.
Previous Review: Freiburg’s way of connection
The main principle of connection is built upon the idea of Golden Gate Connection.( Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).)
The key point of their procedures is a type II Restriction Enzyme, BsmBI enzyme.
Figure 1.4.3 BsmBI recognition site
The main feature of this enzyme is that the recognition sequence is on only one side of cleavage site. It provides the way which can be used to get certain sticky ends with out breaking the whole sequence. The sticky end has 4bp, and it could be designed for combination of multiple sticky ends. That feature is excellent at first, but we cannot ignore its latent shortcomings.
Let’s analyze the example (AA1) provided by Freiburg.
Figure 1.4.4 DNA sequence of AA1 part
The underlined sequence is recognized by BsmBI. Vertical bar(|) is the cutting position. As for this sample, TGAC is one sticky end which can combine with six other sticky ends.
Evaluate seven sticky ends designed by 2012 Freiburg
2012 Freiburg's parts have seven sticky ends:
TGAC,GCTC,CTTG,GCTT,ACTG,CCTG,ACTC
We all know that certain two parts can combine together, under the principle of complementary base pairing. However, is it possible that not totally matched sticky ends can bind together? We found, in fact, the more similar they are, the more possibility that they can form new but incorrect base pairs.
Inspired by BLAST algorithm, we evaluated the similarity of every pair of sticky ends.
Figure 1.4.5 Strict rules score table
The higher score, the higher similarity, and the higher possibility of mismatch.
The table shows that more than 30% of pairs’ score is equal to 3, which means that the possibility of mismatch cannot be neglected.
Even if we employ the relatively loose rule to evaluate the similarity, we can still find that error rates cannot be neglected.
Figure 1.4.6 Loose rules score table
Why not other sticky ends?
The reason why Freiburg used these sticky ends
Failed to contact the original designers of these sticky ends, what we can do is just to find feasible advantages of these combinations.
Let's look at the TALE direpeat unit amino acids sequence:
LTPEQVVAIAS(XX)GGKQALETVQRLLPVLCQAHG(34aa)
The first amino acid is Leu, which is essential for all connection process. There are six different codons for Leu.
UUA,UUG,CUU,CUC,CUA,CUG
The 2012 Freiburg project's sticky ends:
(C)TGAC,GCTC,CTTG,GCTT,ACTG,CCTG,ACTC
The feature of Degeneracy has helped to design seven sticky ends. However, since the codons for identical amino acid are highly similar,
this feature, for experimental scientists, is a double-edged sword.
How to improve the Golden Gate sticky ends? A big Table!
Three key questions need to be answered:
1. Is it possible to find perfect match pair?
2. Can we find a certain number of sticky ends with least mismatch possibility?
3. How to make this sticky-end score table?
Key algorithms derived from BLAST algorithm
Loose rule: Match: 1; Mismatch: 0; Gap: 0
Strict rule: Match: 1; Mismatch: 0; Gap: 1
The sticky end is composed of four bases, which means that we can design 256 types of sticky ends, which are represented as a 256*256 table.
Find target groups of sticky ends
To solve the TALE parts problem, we need find seven sticky ends, and the similarity score of each pair in them are less than or equal to 1.
Figure 1.4.7 Sticky ends choices table
When we select Strict Algorithm to find these ends, it is impossible to find seven sticky ends, that each pair of them has score no more than 1. So we have to select Loose Algorithm.
Convert four-basepair sticky ends to amino acid pairs
We care about whether two amino acids located on our target sequence, rather than the 4bp. So we should convert the sticky ends sequence to amino acid pairs.
Figure 1.4.8 4bp sticky ends convert to Amino acid table
Based on the above table, we are able to calculate the total scores of each combination and find the best one.
Best choice for seven sticky ends on TALE protein
Best combination:
AAAA, AGGG, GTAC, GCTC, TTTT, TCGA, CCCC
Scores Table(Loose rule):
Figure 1.4.9 Best combination score table
Position in TALE amino acids sequence:
Figure 1.4.10 Position in TALE amino acids sequence
Reconstruct DNA sequences
Two main factors to reconstruct DNA sequences:
1.Use the table of best combination and rearrange the sticky ends according to your demand.
2.There are no BsmBI recognition sequence in the reconstruct DNA sequence.
Final DNA Sequence for NEW TALE protein:
1 CTGACCCCGG AACAGGTGGT GGCCATTGCA AGCAACGGTG GTGGCAAGCA GGCCCTGGAG
61 ACAGTCCAAC GGCTGCTTCC GGTTCTGTGT CAGGCCCACG GCCTGACTCC AGAACAAGTG
121 GTTGCTATCG CCAGCCACGA TGGCGGAAAA CAAGCCCTCG AAACCGTGCA GCGCCTGCTT
181 CCGGTGCTGT GTCAGGCCCA CGGGCTCACC CCGGAACAGG TGGTGGCCAT CGCATCTAAC
241 AATGGCGGTA AGCAGGCACT GGAAACAGTG CAGCGCCTGC TTCCGGTCCT GTGTCAGGCT
301 CATGGCCTGA CCCCAGAGCA GGTCGTGGCA ATTGCCTCCA ACATTGGAGG GAAGCAGGCA
361 CTGGAGACCG TGCAGCGGCT GCTGCCGGTG CTGTGTCAGG CCCACGGCTT GACCCCGGAA
421 CAGGTGGTGG CCATCGCCTC CAACGGCGGT GGCAAACAGG CGCTGGAAAC AGTTCAACGC
481 CTCCTTCCGG TCCTGTGCCA GGCCCATGGT CTGACTCCAG AGCAGGTTGT GGCAATTGCA
541 AGCAACATTG GTGGTAAACA AGCTTTGGAA ACCGTCCAGC GCTTGCTGCC AGTACTGTGT
601 CAGGCCCACG GGCTTACCCC GGAACAGGTG GTGGCCATTG CAAGCAACGG TGGTGGCAAG
661 CAGGCCCTGG AGACAGTCCA ACGGCTGCTT CCGGTTCTGT GTCAGGCCCA CGGCCTGACT
721 CCAGAACAAG TGGTTGCTAT CGCCAGCCAC GATGGCGGTA AACAAGCCCT CGAAACCGTG
781 CAGCGCCTGC TTCCGGTGCT CTGTCAGGCC CACGGACTGA CCCCGGAACA GGTGGTGGCC
841 ATCGCCTCCA ACATTGGTGG TAAGCAAGCC CTCGAAACTG TGCAGCGGCT GCTTCCAGTC
901 TTGTGCCAGG CTCACGGCCT GACACCGGAG CAGGTGGTTG CAATCGCGTC TAATATCGGC
961 GGCAAACAGG CACTCGAGAC CGTGCAGCGC TTGCTTCCAG TGCTGTGTCA GGCCCACGGC
1021 CTGACCCCGG AACAGGTGGT GGCCATCGCC TCTAACAATG GCGGCAAACA GGCATTGGAA
1081 ACAGTTCAGC GCCTGCTGCC GGTGTTGTGT CAGGCTCACG GCCTGACTCC GGAGCAGGTT
1141 GTGGCCATCG CAAGCCATGA TGGCGGTAAA CAAGCTCTGG AGACAGTGCA ACGCCTCTTG
1201 CCAGTTTTGT GTCAGGCCCA CGGA
Final Amino acids remain the same:
1 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG
35 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG
69 LTPEQVVAIA SNNGGKQALE TVQRLLPVLC QAHG
103 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG
137 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG
171 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG
205 LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHG
239 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG
273 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG
307 LTPEQVVAIA SNIGGKQALE TVQRLLPVLC QAHG
341 LTPEQVVAIA SNNGGKQALE TVQRLLPVLC QAHG
375 LTPEQVVAIA SHDGGKQALE TVQRLLPVLC QAHG
Corresponding part:
PART-left:
…CTGACCCCGGAGACG
PART1(150bp):
CGTCTCGCCCCGGAACAGGTGGTGGCCATTGCAAGCAACGGTGGTGGCAAGCAGG
CCCTGGAGACAGTCCAACGGCTGCTTCCGGTTCTGTGTCAGGCCCACGGCCTGACT
CCAGAACAAGTGGTTGCTATCGTGGCGGAAAATGAGACG
PART2(219bp):
CGTCTCTAAAACAAGCCCTCGAAACCGTGCAGCGCCTGCTTCCGGTGCTGTGTCAG
GCCCACGGGCTCACCCCGGAACAGGTGGTGGCCATCGCATCTAACAATGGCGGTA
AGCAGGCACTGGAAACAGTGCAGCGCCTGCTTCCGGTCCTGTGTCAGGCTCATGG
CCTGACCCCAGAGCAGGTCGTGGCAATTGCCTCCAACATTGGAGGGCGAGACG
PART3(262bp):
CGTCTCTAGGGAAGCAGGCACTGGAGACCGTGCAGCGGCTGCTGCCGGTGCTGTG
TCAGGCCCACGGCTTGACCCCGGAACAGGTGGTGGCCATCGCCTCCAACGGCGGT
GGCAAACAGGCGCTGGAAACAGTTCAACGCCTCCTTCCGGTCCTGTGCCAGGCCC
ATGGTCTGACTCCAGAGCAGGTTGTGGCAATTGCAAGCAACATTGGTGGTAAACA
AGCTTTGGAAACCGTCCAGCGCTTGCTGCCAGTACGGAGACG
PART4(224bp):
CGTCTCCGTACTGTGTCAGGCCCACGGGCTTACCCCGGAACAGGTGGTGGCCATT
GCAAGCAACGGTGGTGGCAAGCAGGCCCTGGAGACAGTCCAACGGCTGCTTCCGG
TTCTGTGTCAGGCCCACGGCCTGACTCCAGAACAAGTGGTTGCTATCGCCAGCCA
CGATGGCGGTAAACAAGCCCTCGAAACCGTGCAGCGCCTGCTTCCGGTGCTGGGA
GACG
PART5(194bp):
CGTCTCCGCTGTGTCAGGCCCACGGACTGACCCCGGAACAGGTGGTGGCCATCGC
CTCCAACATTGGTGGTAAGCAAGCCCTCGAAACTGTGCAGCGGCTGCTTCCAGTC
TTGTGCCAGGCTCACGGCCTGACACCGGAGCAGGTGGTTGCAATCGCGTCTAATA
TCGGCGGCAAACAGGCACTCGATGAGACG
PART6(249bp):
CGTCTCATCGAGACCGTGCAGCGCTTGCTTCCAGTGCTGTGTCAGGCCCACGGCC
TGACCCCGGAACAGGTGGTGGCCATCGCCTCTAACAATGGCGGCAAACAGGCATT
GGAAACAGTTCAGCGCCTGCTGCCGGTGTTGTGTCAGGCTCACGGCCTGACTCCG
GAGCAGGTTGTGGCCATCGCAAGCCATGATGGCGGTAAACAAGCTCTGGAGACAG
TGCAACGCCTCTTGCCAGTTTTAGAGACG
PART-right:
CGTCTCATTTTGTGTCAGGCCCACGGA...
The recognition sequence of the TALE protein:
TTCGATATCAAGCT
All parts are under artificial synthesis process, so there is few results to present, which can prove our changes are effective. However, with the principle of complementary base pairing, our choice should be better than original version. And if you want our data or use our method to create your own best sticky ends, just contact us!