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− | + | <li style="font-size:22px;"><a class="a_nav">Engineering</a></li> | |
− | + | <li ><a>Overview</a></li> | |
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− | + | <li><a >UAS adding location </a></li> | |
− | + | <li><a >Add UAS </a></li> | |
− | + | <li><a >Multi copy of UAS </a></li> | |
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− | + | <li><a >References</a></li> | |
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− | + | <h2 class="content_text_h2">1. Overview</h2> | |
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− | + | <p id="p1"align="left"> As a team participating in the synthetic Biology Competition, we plan to follow the engineering philosophy of "design-build-test-learn" to carry out our promoter engineering projects. In this process, we apply the “learning by doing” approach. Based on the experience learned from each round of modification, then we continue to design the next round of modification, and finally get the ideal result. We are good at summarizing experience from each modification and some exploring principle from existing research, so as to provide more experience for rational design of promoters in the future. | |
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− | + | <h2 class="content_text_h2">2. UAS adding location</h2> | |
− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 2.1 Design</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p2_1"align="left"> The YeTFaSCo database(<a href="http://yetfasco.ccbr.utoronto.ca/scanSeqs.php">http://yetfasco.ccbr.utoronto.ca/scanSeqs.php</a>) was used to predict the transcription factor binding sites (TFBs) of PPDC1, PSED1L, PALD4. Selected version: 1.02 and resulted in more than 100 putative TFBSs with a matching score higher than 0.75. In order to avoid the incompleteness of using a single database and improve the accuracy of prediction, the YEASTRACT database (<a href="http://www.yeastract.com/formtfsbindingsites.php">http://www.yeastract.com/formtfsbindingsites.php</a>) also used for prediction and comparison. These TFBSs were classified into five groups according to the function (Table 1) | |
− | + | <br><br></p> | |
− | + | <p style="font-size:12px;text-align:center;color:#666666"> | |
− | + | Table 1. Predicted putative TFBs on P<sub>PDC1</sub>, P<sub>SED1L</sub>, P<sub>ALD4</sub> by YeTFaSCo database and YEASTRACT database | |
− | + | </p> | |
− | + | <div class="content_div_img"> | |
− | + | <img src="https://static.igem.org/mediawiki/2021/2/22/T--SCUT-China--engineering_1.png"> | |
− | + | </div> | |
− | + | <p id="p2_2"align="left">According to the characteristics of P<sub>SED1L</sub> and P<sub>ALD4</sub> to control the synthesis of Valencene, which mainly expressed in the later stage of fermentation. We rationally selected the transcription factor binding sites of CAT8p and ADR1p for promoter modification. | |
− | + | </p> | |
− | + | <p id="p2_3"align="left"><br> CAT8p is a Zinc cluster transcriptional activator, which regulate the expression include of most genes in gluconeogenesis, ethanol utilization and glyoxylate cycle<sup> [1]</sup>. CAT8p can bind to carbon source response elements and activate target genes after glucose consumption, the binding motif is 5′-YCCNYTNRKCCG-3′<sup> [2]</sup>. | |
+ | </p> | ||
+ | <p id="p2_4"align="left"><br> ADR1p is a Carbon source-responsive zinc-finger transcription factor, which first identified as a transcription factor activate the transcription of the alcohol dehydrogenase gene ADH2. It also activates genes involved glucose fermentation, glycerol metabolism and fatty acid utilization. The binding motif is 5′-TTGGRG-3′. In addition<sup> [3-4]</sup>, ADR1p and CAT8p may interact when activating the transcription of certain genes<sup> [5]</sup>. | ||
+ | </p> | ||
+ | <p id="p2_5"align="left"><br> In cells, transcription is a process of three-dimensional regulation. The transcription factor binding promoter has a positional effect. When the transcription factor binding site is at a position with high nucleosome affinity, the transcription factor may not be able to bind to the site due to steric hindrance<sup> [6]</sup>. In order to find out which position of PDC1 can make the added UAS (upstream activation sequences) avoid the effect of steric hindrance. The reported UAS1 and the database predicted 100% binding UAS2 were added to the two key positions of PDC1, which can normally bind to transcription factors and activate. UAS1 is the CAT8 binding site of the FBP1 promoter, and UAS2 is the ADR1p binding site predicted to be 100% bound in the ALD4 promoter. Studies have shown that there may be a positive interaction between CAT8p and ADR1p, so we added UAS1 and UAS2 closer to each other. (Figure 1) | ||
+ | </p> | ||
+ | <p id="p2_5_1"align="left"><br> Considering promoter architecture constraints, (a) avoiding a change on other Predicted putative TFBSs for the activation of PDC1 promoter, (b) determining positions for CAT8p and ADR1p binding sites that are close proximity to the core promoter, (c) existing binding sites were replaced with new ones, reduce the changes to the local sequence context. | ||
+ | </p> | ||
+ | <div class="content_div_img"> | ||
+ | <img style="width:70%;"src="https://static.igem.org/mediawiki/2021/1/1a/T--SCUT-China--engineering_3.png"> | ||
+ | </div> | ||
+ | <p style="font-size:12px;text-align:center;color:#666666">Figure 1. The addition of different positions of CAT8 and ADR1</p> | ||
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 2.2 Build</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p2_6"align="left"> Using Overlap PCR method, the sequence at the key position of the PDC1 promoter is replaced. Taking the construction of the M1 mutant strain as an example, the construction method of the M2 strain was the same. Find the CAT8 binding site of FBP1 promoter through the literature and the ADR1 binding site from ALD4 promoter predicted by the database (Table 2).<br><br>Design the binding site motifs on the homology arm, (1) Using primer M1-F/PDC1-R to amplify fragment one, primer M1-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M1 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M1 promoter. Using BamHI and XbaI restriction enzymes to digest the M1 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M1-VS-SAG1t plasmid.<br><br>Then, use donor DNA primers (Table4) to amplify the M1-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M1-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M1-VS-SAG1t expression cassette. | |
− | + | </p> | |
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− | + | <p style="font-size:12px;text-align:center;color:#666666"><br>Table 2. CAT8 and ADR1 binding sites</p> | |
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− | + | <img style="width:46%;"src="https://static.igem.org/mediawiki/2021/4/43/T--SCUT-China--engineering_2.png"> | |
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+ | </div> | ||
− | + | <!-- <p style="font-size:12px;text-align:center;color:#666666"><br>Table 3. Primers of M1 and M2 promoter</p> | |
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− | + | <img style="width:65%;"src="img/Engineering/add_1.png"> | |
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− | + | <p style="font-size:12px;text-align:center;color:#666666"><br>Table 4. Donor DNA primers</p> | |
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− | + | <img style="width:53%;"src="img/Engineering/add_2.png"> | |
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 2.3 Test</h4> | |
− | + | <div class="content_div_text"> | |
+ | <p id="p2_5_2"> | ||
+ | The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 2) | ||
+ | </p> | ||
− | + | <div class="content_div_img"> | |
− | + | <img style="width:110%;"src="https://static.igem.org/mediawiki/2021/8/87/T--SCUT-China--engineering_4.png"> | |
− | + | </div> | |
− | + | <p style="font-size:12px;text-align:center;color:#666666">Figure 2 Fermentation results of M1 and M2 mutant strains. (a) OD<sub>600</sub> of M1 and M2 mutant strain, (b) Valencene yield of M1 and M2 mutant strain. </p> | |
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+ | <p id="p2_5_3"align="left"> | ||
+ | The addition of CAT8 and ADR1 transcription factor binding sites at different positions shows different effects. No significant differences in Valencene production in M1, P<sub>PDC1</sub> were observed. The valencene production of the M2 mutant strain was increased by 18.9% compared to the original strain (P<sub>PDC1</sub>). | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 2.4 Learn</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p2_9"align="left"> In M2, UAS is added about 20 bp upstream of TATA-box located on P<sub>PDC1</sub>, and there are continuous 17 bp A/T near the position of TATA-box, which may cause the structure of this position to be relatively loose and the nucleosome affinity rate Low <sup>[7]</sup>, unable to form a nucleosome structure, transcription factors have a greater probability of binding to this site, and play a role in activating transcription <sup>[8]</sup>. In M1, UAS was added between -393 and -450 located in the upstream region of the PDC1 gene, and it did not work. The possible reason is that it is close to the original activation region, and there is dense transcription factor binding at this site, which hinders the combination of transcription factors to the added UAS. This suggests that we should transform on the basis of M2. | |
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+ | <div id="hrmar"> | ||
+ | <div id="content_part"> | ||
+ | <h2 class="content_text_h2">3. Add UAS</h2> | ||
+ | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 3.1 Design</h4> | ||
+ | <div class="content_div_text"> | ||
+ | <p id="p3_1"align="left"> The YeTFaSCo database and YEASTRACT database were used to predict the CAT8p binding site and ADR1p binding site of SED1L promoter, ALD4 promoter, and select data with a system score of 0.85 or more. According to the results of the first round of transformation, the CAT8p binding site was replaced with the predicted CAT8p binding site of P<sub>SED1L</sub> and P<sub>ALD4</sub> on the basis of M2, but the original ADR1p binding sequence was not changed. (Figure 3) </p> | ||
+ | <div class="content_div_img"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/3/32/T--SCUT-China--engineering_6.png"> | ||
+ | </div> | ||
+ | <p style="font-size:12px;text-align:center;color:#666666"> | ||
+ | Figure 3. the additional add of CAT8p-3 binding site | ||
+ | </p> | ||
+ | </div> | ||
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;"> 3.2 Build</h4> | |
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− | + | Taking the construction of the M3 mutant strain as an example, the construction method of the M4-M7 strain was the same. Find the CAT8 binding site motifs and ADR1 binding site motif predicted by the database (Table 5).<br><br>Design the binding site motifs on the homology arm, (1) Using primer M3-F/PDC1-R to amplify fragment one, primer M3-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M3 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M3 promoter. Using BamHI and XbaI restriction enzymes to digest the M3 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M3-VS-SAG1t plasmid.<br><br>Then, use donor DNA primers (Table 4) to amplify the M3-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M3-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M3-VS-SAG1t expression cassette. | |
− | + | </p> | |
− | + | <p style="font-size:12px;text-align:center;color:#666666"> | |
− | + | Table 5. Predicted CAT8 and ADR1 binding site motif | |
− | + | </p> | |
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− | + | <img style="width:57%;"src="https://static.igem.org/mediawiki/2021/d/d9/T--SCUT-China--engineering_5.png"> | |
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− | + | <!-- <p style="font-size:12px;text-align:center;color:#666666"> | |
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− | + | Table 6. Primers of M3-M7 promoters | |
− | + | </p> | |
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">3.3 Test</h4> | |
+ | <div class="content_div_text"> | ||
+ | <p id="p3_3_0"align="left"> The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 4) </p> | ||
+ | <div class="content_div_img"> | ||
+ | <img style="width:110%;"src="https://static.igem.org/mediawiki/2021/8/84/T--SCUT-China--engineering_7.png"> | ||
+ | </div> | ||
+ | <p style="font-size:12px;text-align:center;color:#666666"> | ||
+ | Figure 4. Fermentation results of M3-M7 mutant strains. (a) OD<sub>600</sub> of M3-M7 mutant strain, (b) Valencene yield of M3-M7 mutant strain. | ||
+ | </p> | ||
− | + | <p id="p3_3"align="left"> | |
− | + | The modified promoters showed different intensities. The Valencene yield of M3, M4, M6, and M7 mutant strains was not significantly different from that of the control strain. But the Valencene yield of the M5 mutant strain was increased by 29.5% (5.58 mg/L) compared to the control strain. </p> | |
− | + | </div> | |
− | + | </div> | |
− | + | <div id="content_part"> | |
− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">3.4 Learn</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p3_4"align="left"> In M5, the CAT8 binding site-3 is derived from the SED1L promoter, which has superior performance compared to the CAT8 binding site from P<sub>FBP1</sub> reported in the article. And we observed that only CAT8 binding site-3 has such an effect, other CAT8 binding sites may be false positive data predicted by the database. This suggests that we can continue to add CAT8 binding site-3 in M5 to increase the expression strength of the promoter. </p> | |
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− | + | <h2 class="content_text_h2">4. Multi copy of UAS</h2> | |
− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">4.1 Design</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p4_1"align="left"> Continue to add CAT8 binding site-3 from P<sub>SED1L</sub> on the basis of M5, add in two locations, the first is added upstream of the original ADR1 binding site, and the second is added to the CAT8 binding site-3 between ADR1 binding site. (Figure 5) </p> | |
− | + | <div class="content_div_img"> | |
− | + | <img src="https://static.igem.org/mediawiki/2021/d/dc/T--SCUT-China--engineering_8.png"> | |
− | + | </div> | |
+ | <p style="font-size:12px;text-align:center;color:#666666"> | ||
+ | Figure 5. The additional add of CAT8p-3 binding site | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="content_part"> | ||
+ | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">4.2 Build</h4> | ||
+ | <div class="content_div_text"> | ||
+ | <p id="p4_2_1"> | ||
+ | Taking the construction of the M8 mutant strain as an example, the construction method of the M9 strain was the same. M10 is constructed on the basis of M8 or M9.<br><br>Design the CAT8 binding site motif on the homology arm, (1) Using primer M8-F/PDC1-R to amplify fragment one, primer M8-R/PDC1-F to amplify fragment two (Table 7), (2) assemble fragment 1 and fragment 2 to obtain M8 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M8 promoter. Using BamHI and XbaI restriction enzymes to digest the M8 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M8-VS-SAG1t plasmid.<br><br>Then, use donor DNA primers (Table 4) to amplify the M8-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M8-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M8-VS-SAG1t expression cassette. | ||
+ | </p> | ||
+ | <!-- <p style="font-size:12px;text-align:center;color:#666666"> | ||
+ | Table 7. Primers of M8 and M9 promoter | ||
+ | </p> | ||
+ | <div class="content_div_img"> | ||
+ | <img style="width:68%;"src="img/Engineering/add_4.png"> | ||
+ | </div> --> | ||
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− | + | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">4.3 Test</h4> | |
− | + | <div class="content_div_text"> | |
− | + | <p id="p4_3_1"> | |
− | + | The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 6) | |
− | + | </p> | |
− | + | <div class="content_div_img"> | |
− | + | <img style="width:110%;"src="https://static.igem.org/mediawiki/2021/0/0e/T--SCUT-China--engineering_9.png"> | |
− | + | </div> | |
− | + | <p style="font-size:12px;text-align:center;color:#666666"> | |
− | + | Figure 6. Figure 6 Fermentation results of M8-M10 mutant strains. (a) OD<sub>600</sub> of M8-M10 mutant strain, (b) Valencene yield of M8-M10 mutant strain. | |
− | + | </p> | |
− | + | <p id="p4_2"align="left"> The Valencene yield of M9 and M10 mutant strains was not significantly improved compared to control strain。However, the Valencene yield of the M8 mutant strain was increased by 27.0% (6.75 mg/L) compared to M5 strain, and 56.6% (6.75 mg/L) compared with the unmodified strain. </p> | |
− | + | </div> | |
− | + | </div> | |
+ | <div id="content_part"> | ||
+ | <h4 class="content_text_h2" style="font-size:18px;margin-top:20px;">4.4 Learn</h4> | ||
+ | <div class="content_div_text"> | ||
+ | <p id="p4_3"align="left"> In M8, CAT8 binding sites-3 was added between the original CAT8 binding sites-3 and ADR1 binding site, which may increase the interaction between CAT8 transcription factor and ADR1 transcription factor. In M9, CAT8 binding site-3 was added to the upstream of the original CAT8 binding site-3, The transcription factor cannot effectively bind to this site, or the add changes the surrounding sequence and may have some negative effects, such as destroying the original binding site of transcription factor with activation. As a result, the Valencene yield of the M9 mutant strain could not be further increased. In M10, the two CAT8 binding sites-3 was added around the ADR1 binding site, which may prevent the binding of ADR1 transcription factors and affect the interaction between CAT8 transcription factors and ADR1 transcription factors. </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <hr class="hrmar" id="content_part_h2" style="visibility:hidden;"> | ||
+ | <div id="hrmar"> | ||
+ | <div id="content_part"> | ||
+ | <h2 class="content_text_h2">5. Summary</h2> | ||
+ | |||
+ | <div class="content_div_text"> | ||
+ | <p id="p4_4"align="left"> The above is the content of the promoter engineering in the wet lab. Through three rounds of "design-build-test-learn", we not only obtained M1-M10 promoters with different strengths, but also summarized the engineering experience from each learning. Our work is summarized as follows. The work presented here, and the promoters designed and engineered through it, represent an attempt to identify yeast regulatory elements that respond to a condition of interest by designing regulatory parts responsive to such a signal––diauxic shift in this case. </p> | ||
+ | <div class="content_div_img"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/d/d4/T--SCUT-China--Engineering-11.png"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <hr class="hrmar" id="content_part_h2" style="visibility:hidden;"> | ||
+ | <div id="hrmar"> | ||
+ | <div id="content_part"> | ||
+ | <h2 class="content_text_h2">6. References</h2> | ||
+ | <div class="content_div_text"> | ||
+ | <p>1. Haurie V, Perrot M, Mini T, Jenö P, Sagliocco F, Boucherie H. The transcriptional activator Cat8p provides a major contribution to the reprogramming of carbon metabolism during the diauxic shift in Saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 2001, 276(1): 76-85. </p> | ||
+ | <p>2. Roth S, Kumme J, Schüller H-J. Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast Saccharomyces cerevisiae[J]. Current Genetics, 2004, 45(3): 121-128. </p> | ||
+ | <p>3. Young ET, Dombek KM, Tachibana C, Ideker T. Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8[J]. The Journal of Biological Chemistry, 2003, 278(28): 26146-26158. </p> | ||
+ | <p>4. Thukral SK, Eisen A, Young ET. Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression[J]. Molecular and Cellular Biology, 1991, 11(3): 1566-1577. </p> | ||
+ | <p>5. Walther K, Schüller HJ. Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae[J]. Microbiology (Reading, England), 2001, 147(Pt 8): 2037-2044. </p> | ||
+ | <p>6. Segal E, Widom J. Poly(dA:dT) tracts: major determinants of nucleosome organization[J]. Current Opinion in Structural Biology, 2009, 19(1): 65-71. </p> | ||
+ | <p>7. Anderson JD, Widom J. Poly(dA-dT) Promoter Elements Increase the Equilibrium Accessibility of Nucleosomal DNA Target Sites[J]. Molecular and Cellular Biology, 2001, 21(11): 3830. </p> | ||
+ | <p>8. Raveh-Sadka T, Levo M, Shabi U, Shany B, Keren L, Lotan-Pompan M, et al. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast[J]. Nature Genetics, 2012, 44(7): 743-750. </p> | ||
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+ | document.getElementById("p1").innerHTML=" 作为一只参与合成生物学竞赛的iGEM队伍 ,我们将按照“设计-建造-测试-学习”的工程理念开展我们的启动子改造工程。在这个过程中,我们采取了<span>“循环学习”</span>的改造思路,基于每一轮改造实验所学习到的经验,接着开展下一轮的改造,以此类推,最终得到理想的改造结果。我们善于从每一次的改造结果中总结经验,善于从已有研究中探寻原理,以便为之后的理性设计提供更多经验。"; | ||
− | + | document.getElementById("p2_1").innerHTML=" 利用YeTFaSCo数据库(http://yetfasco.ccbr.utoronto.ca/scanSeqs.php)预测PPDC1、P<sub>SED1L</sub>、P<sub>ALD4</sub>的转录因子结合位点(TFBs)。所选版本:1.02,结果有100多个假定的TFBSs,匹配分数高于0.75。为了避免使用单个数据库的不完全性,提高预测的准确性,还使用了YEASTRACT数据库(http://www.yeastract.com/formtfsbindingsites.php)进行预测和比较。根据功能将这些TFBSs分为五组。"; | |
− | + | document.getElementById("p2_2").innerHTML=" 根据P<sub>SED1L</sub>和P<sub>ALD4</sub>的特性来控制瓦伦西亚烯的合成,主要表达在发酵后期。我们合理选择了CAT8p和ADR1p的转录因子结合位点进行启动子修饰。"; | |
− | + | document.getElementById("p2_3").innerHTML=" CAT8p是一种锌簇转录激活因子,调节糖异生、乙醇利用和乙醛酸循环[1]中大多数基因的表达。CAT8p与碳源响应元件结合,葡萄糖消耗后激活靶基因,结合基序为5 ' -YCCNYTNRKCCG-3 '[2]。"; | |
− | + | document.getElementById("p2_4").innerHTML=" ADR1p是碳源响应型锌指转录因子,首次被鉴定为激活乙醇脱氢酶基因ADH2转录的转录因子。它还能激活与葡萄糖发酵、甘油代谢和脂肪酸利用有关的基因。结合基序为5 ' -TTGGRG-3 '。此外[3-4],ADR1p和CAT8p可能在激活某些基因[5]的转录时相互作用。"; | |
− | + | document.getElementById("p2_5").innerHTML=" 在细胞中,转录是一个三维调控的过程。转录因子结合启动子具有位置效应。当转录因子结合位点位于核小体亲和度高的位置时,由于空间位阻[6],转录因子可能无法与该位点结合。为了找出PDC1的哪个位置可以使添加的UAS(上游激活序列)避免空间位阻的影响。 报道的UAS1和数据库预测的100%结合UAS2被添加到PDC1的两个关键位置,它可以正常结合转录因子并激活。UAS1是FBP1启动子的CAT8结合位点,UAS2是预测在ALD4启动子中100%结合的ADR1p结合位点。研究表明,CAT8p和ADR1p之间可能存在积极的相互作用,所以我们增加了UAS1和UAS2,两者之间的距离更近。(图1)"; | |
− | + | document.getElementById("p2_5_1").innerHTML=" 考虑到启动子结构的限制,(a)避免PDC1启动子激活的其他预测假定TFBSs的变化,(b)确定靠近核心启动子的CAT8p和ADR1p结合位点的位置,(c)将现有的结合位点替换为新的结合位点,减少对局部序列上下文的更改。"; | |
− | + | document.getElementById("p2_5_2").innerHTML=" 利用重叠PCR方法替换PDC1启动子关键位置的序列。以M1突变株的构建为例,M2突变株的构建方法相同。通过文献找到FBP1启动子的CAT8结合位点和数据库预测的ALD4启动子的ADR1结合位点(表2)。"; | |
− | + | document.getElementById("p2_5_3").innerHTML=" 设计同源臂上的结合位点,(1)利用引物M1- f /PDC1-R扩增片段1,引物M1- r /PDC1-F扩增片段2,(2)组装片段1和片段2得到M1启动子,(3)利用引物PDC1-F/PDC1-R扩增M1启动子。利用BamHI和XbaI酶切酶消化M1启动子和YEp181-PDC1p-VS-SAG1t载体,37℃反应2h。反应结束后,将启动子与载体进行纯化,16℃过夜连接。转化大肠杆菌DH5α,选择正确的转化子,提取YEp181-M1-VS-SAG1t质粒。"; | |
+ | document.getElementById("p2_6").innerHTML=" 然后,使用donor DNA引物(表4)扩增M1-VS-SAG1t表达盒,在LEU2位点旁边有一个同源臂。将构建的gRNA表达质粒和M1-VS-SAG1t表达盒转化到能表达Cas9蛋白的酵母细胞中,采用营养缺陷培养基选择和菌落PCR验证的方法,获得成功敲入M1-VS-SAG1t表达盒的菌株。"; | ||
+ | document.getElementById("p2_9").innerHTML=" 在M2的改造中,UAS被添加在偏向于P<sub>PDC1</sub>上游,大约20 bp的位置,有连续17 bp / T着重的位置附近,这可能导致这个位置的结构相对松散,[7]核小体亲和率低,无法形成核小体结构,转录因子与该位点结合的可能性较大,并在激活转录[8]中发挥作用。在M1改造中,在PDC1基因上游区域的-393 ~ -450之间添加了UAS,未起作用。可能的原因是该位点靠近原始激活区,且有密集的转录因子结合,阻碍了转录因子与添加的UAS的结合。这意味着我们应该在M2的基础上进行进一步改造。"; | ||
+ | document.getElementById("p3_1").innerHTML=" 利用YeTFaSCo数据库和YEASTRACT数据库预测SED1L启动子、ALD4启动子的CAT8p结合位点和ADR1p结合位点,并选择系统评分为0.85及以上的数据。根据第一轮转化的结果,在M2的基础上,将CAT8p结合位点替换为预测的P<sub>SED1L</sub>和P<sub>ALD4</sub>的CAT8p结合位点,但原ADR1p结合序列没有改变。(图3)"; | ||
+ | document.getElementById("p3_2_1").innerHTML=" 以M3突变株的构建为例,M4-M7菌株的构建方法相同。查找数据库预测的CAT8结合位点motif和ADR1结合位点motif(表5)。<br><br>设计同源臂上的结合位点,(1)利用引物M3- f /PDC1-R扩增片段1,引物M3- r /PDC1-F扩增片段2,(2)组装片段1和片段2得到M3启动子,(3)利用引物PDC1-F/PDC1-R扩增M3启动子。利用BamHI和XbaI酶切酶消化M3启动子和YEp181-PDC1p-VS-SAG1t载体,37℃反应2h。反应结束后,将启动子与载体进行纯化,16℃过夜连接。转化大肠杆菌DH5α,选择正确的转化子,提取YEp181-M3-VS-SAG1t质粒。<br><br>然后,使用donor DNA引物(表4)扩增M3-VS-SAG1t表达盒,在LEU2位点旁边有一个同源臂。将构建的gRNA表达质粒和M3-VS-SAG1t表达盒转化到能表达Cas9蛋白的酵母细胞中,采用营养缺陷培养基选择和菌落PCR验证的方法,获得成功敲入M3-VS-SAG1t表达盒的菌株。"; | ||
+ | |||
+ | document.getElementById("p3_3_0").innerHTML=" 该菌株接种到10 ml发酵液中,控制发酵液初始OD600为0.05,置于30、220 rpm的摇瓶中孵育64小时。摇瓶发酵64小时后,用气相色谱法检测瓦伦西亚烯的浓度。(图4)"; | ||
+ | document.getElementById("p3_3").innerHTML=" 这轮改造后的的启动子表现出不同的强度。M3、M4、M6和M7突变株的瓦伦西亚烯产量与对照无显著差异。而M5突变株的瓦伦西亚烯产量比对照株提高29.5% (5.58 mg/L)。"; | ||
+ | document.getElementById("p3_4").innerHTML=" 在M5中,CAT8结合位点-3来源于SED1L启动子,与本文报道的来自P<sub>FBP1</sub>的CAT8结合位点相比,其性能更优越。并且我们观察到只有CAT8结合位点-3具有这种作用,其他的CAT8结合位点可能是数据库预测的假阳性数据。这表明我们可以继续在M5中添加CAT8结合位点-3,以提高启动子的表达强度。"; | ||
+ | document.getElementById("p4_1").innerHTML=" 在M5的基础上继续从P<sub>SED1L</sub>添加CAT8结合位点-3,在两个位点上添加,第一个位点添加在原ADR1结合位点的上游,第二个位点添加在ADR1结合位点之间的CAT8结合位点-3。(图5)"; | ||
+ | document.getElementById("p4_2_1").innerHTML=" 以M8突变株的构建为例,M9菌株的构建方法与之相同。M10是在M8或M9的基础上构建的。在同源臂上设计CAT8结合位点motif,(1)利用引物M8- f /PDC1-R扩增片段1,引物M8- r /PDC1-F扩增片段2(表7),(2)组装片段1和片段2得到M8启动子,(3)利用引物PDC1-F/PDC1-R扩增M8启动子。利用BamHI和XbaI酶切酶消化M8启动子和YEp181-PDC1p-VS-SAG1t载体,在37℃反应2h。反应结束后,启动子和载体被纯化并在16c连接过夜。转化大肠杆菌DH5α,选择正确的转化子,提取YEp181-M8-VS-SAG1t质粒。然后,使用donor DNA引物(表4)扩增M8-VS-SAG1t表达盒,在LEU2位点旁边有一个同源臂。将构建的gRNA表达质粒和M8-VS-SAG1t表达盒转化到能表达Cas9蛋白的酵母细胞中,采用营养缺陷培养基选择和菌落PCR验证的方法,获得成功敲入M8-VS-SAG1t表达盒的菌株。"; | ||
+ | document.getElementById("p4_3_1").innerHTML=" 将该菌株接种到10 ml发酵液中,控制发酵液初始OD600为0.05,置于30、220 rpm的摇瓶中孵育64小时。摇瓶发酵64小时后,用气相色谱法检测瓦伦西亚烯的浓度。(图6)"; | ||
+ | document.getElementById("p4_2").innerHTML=" M9和M10突变株的瓦伦西亚烯产量与对照相比没有显著提高。M8突变株的瓦伦烯产量比M5突变株提高了27.0% (6.75 mg/L),比未突变株提高了56.6% (6.75 mg/L)。"; | ||
+ | document.getElementById("p4_3").innerHTML=" 在M8的改造中,原有的CAT8结合位点-3与ADR1结合位点之间增加了CAT8结合位点-3,这可能增加了CAT8转录因子与ADR1转录因子之间的相互作用。在M9的改造中,在原CAT8结合位点-3的上游添加了CAT8结合位点-3,导致转录因子不能有效地与该位点结合,或者添加改变了周围的序列,可能会产生一些负面影响,如激活后破坏了转录因子原有的结合位点。结果表明,M9突变株的瓦伦西亚烯产量不能进一步提高。在M10的改造中,在ADR1结合位点周围添加了两个CAT8结合位点-3,这可能会阻止ADR1转录因子的结合,影响CAT8转录因子与ADR1转录因子的相互作用。"; | ||
+ | |||
+ | }else{ | ||
+ | document.getElementById("p1").innerHTML=" As a team participating in the synthetic Biology Competition, we plan to follow the engineering philosophy of "design-build-test-learn" to carry out our promoter engineering projects. In this process, we apply the “learning by doing” approach. Based on the experience learned from each round of modification, then we continue to design the next round of modification, and finally get the ideal result. We are good at summarizing experience from each modification and some exploring principle from existing research, so as to provide more experience for rational design of promoters in the future."; | ||
+ | document.getElementById("p2_1").innerHTML=" The YeTFaSCo database were used to predict the transcription factor binding sites (TFBS) of P<SUB>PDC1</SUB>, P<SUB>SED1L</SUB>, P<sub>ALD4</sub>. Selected Version: 1.02 and resulted in more than 100 putative TFBSs with a matching score higher than 0.75. These TFBSs were classified into five groups according to the function (Table 1)"; | ||
+ | document.getElementById("p2_2").innerHTML=" According to the characteristics of P<sub>SED1L</sub> and P<sub>ALD4</sub> to control the synthesis of Valencene, which mainly expressed in the later stage of fermentation. We rationally selected the transcription factor binding sites of CAT8p and ADR1p for promoter modification."; | ||
+ | document.getElementById("p2_3").innerHTML=" CAT8p is a Zinc cluster transcriptional activator, which regulate the expression include of most genes in gluconeogenesis, ethanol utilization and glyoxylate cycle<sup> [1]</sup>. CAT8p can bind to carbon source response elements and activate target genes after glucose consumption, the binding motif is 5′-YCCNYTNRKCCG-3′<sup> [2]</sup>. "; | ||
+ | document.getElementById("p2_4").innerHTML=" ADR1p is a Carbon source-responsive zinc-finger transcription factor, which first identified as a transcription factor activate the transcription of the alcohol dehydrogenase gene ADH2. It also activates genes involved glucose fermentation, glycerol metabolism and fatty acid utilization. The binding motif is 5′-TTGGRG-3′. In addition<sup> [3-4]</sup>, ADR1p and CAT8p may interact when activating the transcription of certain genes<sup> [5]</sup>."; | ||
+ | document.getElementById("p2_5").innerHTML=" In cells, transcription is a process of three-dimensional regulation. The transcription factor binding promoter has a positional effect. When the transcription factor binding site is at a position with high nucleosome affinity, the transcription factor may not be able to bind to the site due to steric hindrance<sup> [6]</sup>. In order to find out which position of PDC1 can make the added UAS (upstream activation sequences) avoid the effect of steric hindrance. The reported UAS1 and the database predicted 100% binding UAS2 were added to the two key positions of PDC1, which can normally bind to transcription factors and activate. UAS1 is the CAT8 binding site of the FBP1 promoter, and UAS2 is the ADR1p binding site predicted to be 100% bound in the ALD4 promoter. Studies have shown that there may be a positive interaction between CAT8p and ADR1p, so we added UAS1 and UAS2 closer to each other. (Figure 1)"; | ||
+ | document.getElementById("p2_5_1").innerHTML=" Considering promoter architecture constraints, (a) avoiding a change on other Predicted putative TFBSs for the activation of PDC1 promoter, (b) determining positions for CAT8p and ADR1p binding sites that are close proximity to the core promoter, (c) existing binding sites were replaced with new ones, reduce the changes to the local sequence context."; | ||
+ | document.getElementById("p2_5_2").innerHTML=" The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 2)"; | ||
+ | document.getElementById("p2_5_3").innerHTML=" The addition of CAT8 and ADR1 transcription factor binding sites at different positions shows different effects. No significant differences in Valencene production in M1, P<sub>PDC1</sub> were observed. The valencene production of the M2 mutant strain was increased by 18.9% compared to the original strain (P<sub>PDC1</sub>)."; | ||
+ | |||
+ | |||
+ | document.getElementById("p2_6").innerHTML=" Using Overlap PCR method, the sequence at the key position of the PDC1 promoter is replaced. Taking the construction of the M1 mutant strain as an example, the construction method of the M2 strain was the same. Find the CAT8 binding site of FBP1 promoter through the literature and the ADR1 binding site from ALD4 promoter predicted by the database (Table 2).<br><br>Design the binding site motifs on the homology arm, (1) Using primer M1-F/PDC1-R to amplify fragment one, primer M1-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M1 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M1 promoter. Using BamHI and XbaI restriction enzymes to digest the M1 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M1-VS-SAG1t plasmid.<br><br></br>Then, use donor DNA primers (Table4) to amplify the M1-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M1-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M1-VS-SAG1t expression cassette."; | ||
+ | document.getElementById("p2_9").innerHTML=" In M2, UAS is added about 20 bp upstream of TATA-box located on P<sub>PDC1</sub>, and there are continuous 17 bp A/T near the position of TATA-box, which may cause the structure of this position to be relatively loose and the nucleosome affinity rate Low <sup>[7]</sup>, unable to form a nucleosome structure, transcription factors have a greater probability of binding to this site, and play a role in activating transcription <sup>[8]</sup>. In M1, UAS was added between -393 and -450 located in the upstream region of the PDC1 gene, and it did not work. The possible reason is that it is close to the original activation region, and there is dense transcription factor binding at this site, which hinders the combination of transcription factors to the added UAS. This suggests that we should transform on the basis of M2."; | ||
+ | document.getElementById("p3_1").innerHTML=" The YeTFaSCo database and YEASTRACT database were used to predict the CAT8p binding site and ADR1p binding site of SED1L promoter, ALD4 promoter, and select data with a system score of 0.85 or more. According to the results of the first round of transformation, the CAT8p binding site was replaced with the predicted CAT8p binding site of P<sub>SED1L</sub> and P<sub>ALD4</sub> on the basis of M2, but the original ADR1p binding sequence was not changed. (Figure 3)"; | ||
+ | document.getElementById("p3_2_1").innerHTML=" Taking the construction of the M3 mutant strain as an example, the construction method of the M4-M7 strain was the same. Find the CAT8 binding site motifs and ADR1 binding site motif predicted by the database (Table 5).<br><br>Design the binding site motifs on the homology arm, (1) Using primer M3-F/PDC1-R to amplify fragment one, primer M3-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M3 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M3 promoter. Using BamHI and XbaI restriction enzymes to digest the M3 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M3-VS-SAG1t plasmid.<br><br>Then, use donor DNA primers (Table 4) to amplify the M3-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M3-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M3-VS-SAG1t expression cassette."; | ||
+ | |||
+ | document.getElementById("p3_3_0").innerHTML=" The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 4)"; | ||
+ | document.getElementById("p3_3").innerHTML=" The modified promoters showed different intensities. The Valencene yield of M3, M4, M6, and M7 mutant strains was not significantly different from that of the control strain. But the Valencene yield of the M5 mutant strain was increased by 29.5% (5.58 mg/L) compared to the control strain."; | ||
+ | document.getElementById("p3_4").innerHTML=" In M5, the CAT8 binding site-3 is derived from the SED1L promoter, which has superior performance compared to the CAT8 binding site from P<sub>FBP1</sub> reported in the article. And we observed that only CAT8 binding site-3 has such an effect, other CAT8 binding sites may be false positive data predicted by the database. This suggests that we can continue to add CAT8 binding site-3 in M5 to increase the expression strength of the promoter."; | ||
+ | document.getElementById("p4_1").innerHTML=" Continue to add CAT8 binding site-3 from P<sub>SED1L</sub> on the basis of M5, add in two locations, the first is added upstream of the original ADR1 binding site, and the second is added to the CAT8 binding site-3 between ADR1 binding site. (Figure 5)"; | ||
+ | document.getElementById("p4_2_1").innerHTML=" Taking the construction of the M8 mutant strain as an example, the construction method of the M9 strain was the same. M10 is constructed on the basis of M8 or M9.<br><br>Design the CAT8 binding site motif on the homology arm, (1) Using primer M8-F/PDC1-R to amplify fragment one, primer M8-R/PDC1-F to amplify fragment two (Table 7), (2) assemble fragment 1 and fragment 2 to obtain M8 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M8 promoter. Using BamHI and XbaI restriction enzymes to digest the M8 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M8-VS-SAG1t plasmid.<br><br>Then, use donor DNA primers (Table 4) to amplify the M8-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M8-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M8-VS-SAG1t expression cassette."; | ||
+ | |||
+ | document.getElementById("p4_3_1").innerHTML=" The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 6)"; | ||
+ | document.getElementById("p4_2").innerHTML=" The Valencene yield of M9 and M10 mutant strains was not significantly improved compared to control strain。However, the Valencene yield of the M8 mutant strain was increased by 27.0% (6.75 mg/L) compared to M5 strain, and 56.6% (6.75 mg/L) compared with the unmodified strain."; | ||
+ | document.getElementById("p4_3").innerHTML=" In M8, CAT8 binding sites-3 was added between the original CAT8 binding sites-3 and ADR1 binding site, which may increase the interaction between CAT8 transcription factor and ADR1 transcription factor. In M9, CAT8 binding site-3 was added to the upstream of the original CAT8 binding site-3, The transcription factor cannot effectively bind to this site, or the add changes the surrounding sequence and may have some negative effects, such as destroying the original binding site of transcription factor with activation. As a result, the Valencene yield of the M9 mutant strain could not be further increased. In M10, the two CAT8 binding sites-3 was added around the ADR1 binding site, which may prevent the binding of ADR1 transcription factors and affect the interaction between CAT8 transcription factors and ADR1 transcription factors."; | ||
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Revision as of 21:12, 21 October 2021
1. Overview
As a team participating in the synthetic Biology Competition, we plan to follow the engineering philosophy of "design-build-test-learn" to carry out our promoter engineering projects. In this process, we apply the “learning by doing” approach. Based on the experience learned from each round of modification, then we continue to design the next round of modification, and finally get the ideal result. We are good at summarizing experience from each modification and some exploring principle from existing research, so as to provide more experience for rational design of promoters in the future.
2. UAS adding location
2.1 Design
The YeTFaSCo database(http://yetfasco.ccbr.utoronto.ca/scanSeqs.php) was used to predict the transcription factor binding sites (TFBs) of PPDC1, PSED1L, PALD4. Selected version: 1.02 and resulted in more than 100 putative TFBSs with a matching score higher than 0.75. In order to avoid the incompleteness of using a single database and improve the accuracy of prediction, the YEASTRACT database (http://www.yeastract.com/formtfsbindingsites.php) also used for prediction and comparison. These TFBSs were classified into five groups according to the function (Table 1)
Table 1. Predicted putative TFBs on PPDC1, PSED1L, PALD4 by YeTFaSCo database and YEASTRACT database
According to the characteristics of PSED1L and PALD4 to control the synthesis of Valencene, which mainly expressed in the later stage of fermentation. We rationally selected the transcription factor binding sites of CAT8p and ADR1p for promoter modification.
CAT8p is a Zinc cluster transcriptional activator, which regulate the expression include of most genes in gluconeogenesis, ethanol utilization and glyoxylate cycle [1]. CAT8p can bind to carbon source response elements and activate target genes after glucose consumption, the binding motif is 5′-YCCNYTNRKCCG-3′ [2].
ADR1p is a Carbon source-responsive zinc-finger transcription factor, which first identified as a transcription factor activate the transcription of the alcohol dehydrogenase gene ADH2. It also activates genes involved glucose fermentation, glycerol metabolism and fatty acid utilization. The binding motif is 5′-TTGGRG-3′. In addition [3-4], ADR1p and CAT8p may interact when activating the transcription of certain genes [5].
In cells, transcription is a process of three-dimensional regulation. The transcription factor binding promoter has a positional effect. When the transcription factor binding site is at a position with high nucleosome affinity, the transcription factor may not be able to bind to the site due to steric hindrance [6]. In order to find out which position of PDC1 can make the added UAS (upstream activation sequences) avoid the effect of steric hindrance. The reported UAS1 and the database predicted 100% binding UAS2 were added to the two key positions of PDC1, which can normally bind to transcription factors and activate. UAS1 is the CAT8 binding site of the FBP1 promoter, and UAS2 is the ADR1p binding site predicted to be 100% bound in the ALD4 promoter. Studies have shown that there may be a positive interaction between CAT8p and ADR1p, so we added UAS1 and UAS2 closer to each other. (Figure 1)
Considering promoter architecture constraints, (a) avoiding a change on other Predicted putative TFBSs for the activation of PDC1 promoter, (b) determining positions for CAT8p and ADR1p binding sites that are close proximity to the core promoter, (c) existing binding sites were replaced with new ones, reduce the changes to the local sequence context.
Figure 1. The addition of different positions of CAT8 and ADR1
2.2 Build
Using Overlap PCR method, the sequence at the key position of the PDC1 promoter is replaced. Taking the construction of the M1 mutant strain as an example, the construction method of the M2 strain was the same. Find the CAT8 binding site of FBP1 promoter through the literature and the ADR1 binding site from ALD4 promoter predicted by the database (Table 2).
Design the binding site motifs on the homology arm, (1) Using primer M1-F/PDC1-R to amplify fragment one, primer M1-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M1 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M1 promoter. Using BamHI and XbaI restriction enzymes to digest the M1 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M1-VS-SAG1t plasmid.
Then, use donor DNA primers (Table4) to amplify the M1-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M1-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M1-VS-SAG1t expression cassette.
Table 2. CAT8 and ADR1 binding sites
2.3 Test
The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 2)
Figure 2 Fermentation results of M1 and M2 mutant strains. (a) OD600 of M1 and M2 mutant strain, (b) Valencene yield of M1 and M2 mutant strain.
The addition of CAT8 and ADR1 transcription factor binding sites at different positions shows different effects. No significant differences in Valencene production in M1, PPDC1 were observed. The valencene production of the M2 mutant strain was increased by 18.9% compared to the original strain (PPDC1).
2.4 Learn
In M2, UAS is added about 20 bp upstream of TATA-box located on PPDC1, and there are continuous 17 bp A/T near the position of TATA-box, which may cause the structure of this position to be relatively loose and the nucleosome affinity rate Low [7], unable to form a nucleosome structure, transcription factors have a greater probability of binding to this site, and play a role in activating transcription [8]. In M1, UAS was added between -393 and -450 located in the upstream region of the PDC1 gene, and it did not work. The possible reason is that it is close to the original activation region, and there is dense transcription factor binding at this site, which hinders the combination of transcription factors to the added UAS. This suggests that we should transform on the basis of M2.
3. Add UAS
3.1 Design
The YeTFaSCo database and YEASTRACT database were used to predict the CAT8p binding site and ADR1p binding site of SED1L promoter, ALD4 promoter, and select data with a system score of 0.85 or more. According to the results of the first round of transformation, the CAT8p binding site was replaced with the predicted CAT8p binding site of PSED1L and PALD4 on the basis of M2, but the original ADR1p binding sequence was not changed. (Figure 3)
Figure 3. the additional add of CAT8p-3 binding site
3.2 Build
Taking the construction of the M3 mutant strain as an example, the construction method of the M4-M7 strain was the same. Find the CAT8 binding site motifs and ADR1 binding site motif predicted by the database (Table 5).
Design the binding site motifs on the homology arm, (1) Using primer M3-F/PDC1-R to amplify fragment one, primer M3-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M3 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M3 promoter. Using BamHI and XbaI restriction enzymes to digest the M3 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M3-VS-SAG1t plasmid.
Then, use donor DNA primers (Table 4) to amplify the M3-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M3-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M3-VS-SAG1t expression cassette.
Table 5. Predicted CAT8 and ADR1 binding site motif
3.3 Test
The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 4)
Figure 4. Fermentation results of M3-M7 mutant strains. (a) OD600 of M3-M7 mutant strain, (b) Valencene yield of M3-M7 mutant strain.
The modified promoters showed different intensities. The Valencene yield of M3, M4, M6, and M7 mutant strains was not significantly different from that of the control strain. But the Valencene yield of the M5 mutant strain was increased by 29.5% (5.58 mg/L) compared to the control strain.
3.4 Learn
In M5, the CAT8 binding site-3 is derived from the SED1L promoter, which has superior performance compared to the CAT8 binding site from PFBP1 reported in the article. And we observed that only CAT8 binding site-3 has such an effect, other CAT8 binding sites may be false positive data predicted by the database. This suggests that we can continue to add CAT8 binding site-3 in M5 to increase the expression strength of the promoter.
4. Multi copy of UAS
4.1 Design
Continue to add CAT8 binding site-3 from PSED1L on the basis of M5, add in two locations, the first is added upstream of the original ADR1 binding site, and the second is added to the CAT8 binding site-3 between ADR1 binding site. (Figure 5)
Figure 5. The additional add of CAT8p-3 binding site
4.2 Build
Taking the construction of the M8 mutant strain as an example, the construction method of the M9 strain was the same. M10 is constructed on the basis of M8 or M9.
Design the CAT8 binding site motif on the homology arm, (1) Using primer M8-F/PDC1-R to amplify fragment one, primer M8-R/PDC1-F to amplify fragment two (Table 7), (2) assemble fragment 1 and fragment 2 to obtain M8 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M8 promoter. Using BamHI and XbaI restriction enzymes to digest the M8 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M8-VS-SAG1t plasmid.
Then, use donor DNA primers (Table 4) to amplify the M8-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M8-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M8-VS-SAG1t expression cassette.
4.3 Test
The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 6)
Figure 6. Figure 6 Fermentation results of M8-M10 mutant strains. (a) OD600 of M8-M10 mutant strain, (b) Valencene yield of M8-M10 mutant strain.
The Valencene yield of M9 and M10 mutant strains was not significantly improved compared to control strain。However, the Valencene yield of the M8 mutant strain was increased by 27.0% (6.75 mg/L) compared to M5 strain, and 56.6% (6.75 mg/L) compared with the unmodified strain.
4.4 Learn
In M8, CAT8 binding sites-3 was added between the original CAT8 binding sites-3 and ADR1 binding site, which may increase the interaction between CAT8 transcription factor and ADR1 transcription factor. In M9, CAT8 binding site-3 was added to the upstream of the original CAT8 binding site-3, The transcription factor cannot effectively bind to this site, or the add changes the surrounding sequence and may have some negative effects, such as destroying the original binding site of transcription factor with activation. As a result, the Valencene yield of the M9 mutant strain could not be further increased. In M10, the two CAT8 binding sites-3 was added around the ADR1 binding site, which may prevent the binding of ADR1 transcription factors and affect the interaction between CAT8 transcription factors and ADR1 transcription factors.
5. Summary
The above is the content of the promoter engineering in the wet lab. Through three rounds of "design-build-test-learn", we not only obtained M1-M10 promoters with different strengths, but also summarized the engineering experience from each learning. Our work is summarized as follows. The work presented here, and the promoters designed and engineered through it, represent an attempt to identify yeast regulatory elements that respond to a condition of interest by designing regulatory parts responsive to such a signal––diauxic shift in this case.
6. References
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