In order to improve this situation, we have explored the Tammy M Hsu’s and 2013 IGEM_Berkeley’s study1 and produced a system to express indigo in E. coli with a protecting group strategy (Figure 1).
![](https://static.igem.org/mediawiki/2018/5/55/T--SCU-China--indigo1.jpg)
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We have successfully constructed the reporter plasmid and verified that it could function well (T7 promoter-RFP-J23100-GFP): | We have successfully constructed the reporter plasmid and verified that it could function well (T7 promoter-RFP-J23100-GFP): | ||
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T7 promoter-RFP-J23100-GFP-pSB1C3 has been successfully constructed. The normal function of the GFP and mRFP1 have been verified. | T7 promoter-RFP-J23100-GFP-pSB1C3 has been successfully constructed. The normal function of the GFP and mRFP1 have been verified. | ||
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The electrophoretogram of ligation result is shown as follows. | The electrophoretogram of ligation result is shown as follows. | ||
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<div class="subsubtitle">The Functional Test</div> | <div class="subsubtitle">The Functional Test</div> | ||
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The successful ligation of T7-gRNA (J23)-pSB1C3 backbone and J23100-GFP inserts using 3A assembly is shown as follows. | The successful ligation of T7-gRNA (J23)-pSB1C3 backbone and J23100-GFP inserts using 3A assembly is shown as follows. | ||
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We tried to apply Red/ET4 system, which comes from Lambda bacteriophage, and RecA protein 5,6, which could improve the stability of single strand DNA, to assist the | We tried to apply Red/ET4 system, which comes from Lambda bacteriophage, and RecA protein 5,6, which could improve the stability of single strand DNA, to assist the | ||
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HR process. Besides, two plasmids system or two step method will work at low efficiency and is time-consuming, so we try to combine all parts into one big plasmid which could shorten the period of workflow. There should be no plasmid or antibiotic gene remaining in bacteria after genome editing, therefore, we used pSC101 temperature sensitive origin of replication7, the plasmid containing which would replicate at a low efficiency under 37℃ and finally loses after 1 day’s incubation. Considering the cytotoxicity of Cas9 protein, and the controllability of knock in system, we used arabinose (Ara) promoter to control the expression of Cas9 protein and recombination related proteins, because the leaky expression of Cas9 protein could not only slow down the grow of bacteria, but also decrease the efficiency of transformation. Providing glucose only should inhibit gene transcription. While providing arabinose only, the downstream expression system could be active. | HR process. Besides, two plasmids system or two step method will work at low efficiency and is time-consuming, so we try to combine all parts into one big plasmid which could shorten the period of workflow. There should be no plasmid or antibiotic gene remaining in bacteria after genome editing, therefore, we used pSC101 temperature sensitive origin of replication7, the plasmid containing which would replicate at a low efficiency under 37℃ and finally loses after 1 day’s incubation. Considering the cytotoxicity of Cas9 protein, and the controllability of knock in system, we used arabinose (Ara) promoter to control the expression of Cas9 protein and recombination related proteins, because the leaky expression of Cas9 protein could not only slow down the grow of bacteria, but also decrease the efficiency of transformation. Providing glucose only should inhibit gene transcription. While providing arabinose only, the downstream expression system could be active. | ||
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In knock in part, we used Modified Gibson Assembly to construct the donor DNA and backbone which is prepared by high speed PCR enzymes (Primer STAR○R GXL). To get a good electroporation transformation efficiency, we purify the reaction product by column (E.Z.N.A.® Plasmid Mini Kit I). After SOC culture at 30℃ for 1h, we plated the electro transformation production on plate with 1%(w/v) glucose, and 50ng/ul kanamycin. Finally, we selected several colonies for screening and re-plated them. | In knock in part, we used Modified Gibson Assembly to construct the donor DNA and backbone which is prepared by high speed PCR enzymes (Primer STAR○R GXL). To get a good electroporation transformation efficiency, we purify the reaction product by column (E.Z.N.A.® Plasmid Mini Kit I). After SOC culture at 30℃ for 1h, we plated the electro transformation production on plate with 1%(w/v) glucose, and 50ng/ul kanamycin. Finally, we selected several colonies for screening and re-plated them. | ||
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After successfully plasmid construction, we selected new single colony for amplification and cultured colony with solid L-Ara (1%) media overnight and selected colony randomly for colony PCR which we get 2 in 14 has knock-in feature. | After successfully plasmid construction, we selected new single colony for amplification and cultured colony with solid L-Ara (1%) media overnight and selected colony randomly for colony PCR which we get 2 in 14 has knock-in feature. | ||
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Then we re-plated the colony on plate. Interestingly not all the colony turn red at the same time. | Then we re-plated the colony on plate. Interestingly not all the colony turn red at the same time. | ||
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In order to test the elimination of plasmid, we cultured the E.coli on 37℃ for 1 day, and then plated it on plasmid on K+ plate compared by those without elimination. Those still have plasmid (right) survived better than the left. And the plasmid purification from 6 ml colony culture shows no plasmid remaining. | In order to test the elimination of plasmid, we cultured the E.coli on 37℃ for 1 day, and then plated it on plasmid on K+ plate compared by those without elimination. Those still have plasmid (right) survived better than the left. And the plasmid purification from 6 ml colony culture shows no plasmid remaining. | ||
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<div class="subsubtitle">4.2 Donor part preparing</div> | <div class="subsubtitle">4.2 Donor part preparing</div> | ||
At the same time, we design the universal primers so that we can use these primers to amplify any sequence on between prefix and suffix. Thus, we used these primers to amplified the GFP, RFP+GFP, dCas9 and dCas9 with GFP for future cloning. | At the same time, we design the universal primers so that we can use these primers to amplify any sequence on between prefix and suffix. Thus, we used these primers to amplified the GFP, RFP+GFP, dCas9 and dCas9 with GFP for future cloning. | ||
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In this part, we successfully verified the knock in function of our plasmid, and test the Golden Gate assembly plasmid construction. Besides here are some drawbacks of this methods.1 It’s hard to transformation efficiency, for with only the colony PCR it could be a low efficiency for colony selection when construction plasmid. 2 The periodic expression of genome may result in the desynchrony expression of RFP on genome. 3 the exogenous gene in Bacteria genome may result in long fragment homologous recombination within genome range. We only get positive result for the first few days. After several days culture after losing its plasmid, the same site colony will smear or abnormal length fragment will rise (see notebook) which may suggest the HR in genome range. | In this part, we successfully verified the knock in function of our plasmid, and test the Golden Gate assembly plasmid construction. Besides here are some drawbacks of this methods.1 It’s hard to transformation efficiency, for with only the colony PCR it could be a low efficiency for colony selection when construction plasmid. 2 The periodic expression of genome may result in the desynchrony expression of RFP on genome. 3 the exogenous gene in Bacteria genome may result in long fragment homologous recombination within genome range. We only get positive result for the first few days. After several days culture after losing its plasmid, the same site colony will smear or abnormal length fragment will rise (see notebook) which may suggest the HR in genome range. | ||