Team:Tsinghua-E/Part3

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<h2>Part 3: THU-E Sweet Pressure Part</h2> <br />
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<p>  This is a plasmid providing selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype by glucose utilization.The selection pressure was based on the tryptophan dependent maltose hydrolase expression which functioned in a maltose-sole carbon source culture condition. It is achieved by cloning <em>E. Coli</em> maltose hydrolase gene (<em>malQ</em>) downstream of our previously constructed tryptophan biosensor which is controlled by the strict araBAD promoter in a<em>malQ</em>deletion strain E. Coli JW3379.A random ribosome binding sequence (RBS) library upstream of <em>malQ</em>were constructed. 48 strains from the library were selectedbased onthe growth difference between maltose and  glucose M9 minimal culture condition. Strain 3-4 with the RBS sequence“AAAAAAATGTGCT”  was selected and named after sweet pressure part strain. <br />
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  The  performance of the sweet pressure part strain 3-4 was further measured in a tryptophan addition fashion. The colony from LB agar plate was picked up and  cultured in M9 medium with 8g/L glucose for overnight. On the next day, seed  medium was transferred to fresh M9 medium in 24 well plate with2, 4, 6 and 8g/L maltose as single carbon source, respectively. Initial OD600 was set  as 0.16. When OD600 of the medium reached 0.6, 0, 0.5 and 1g/L L-arabinose, 0 and 1mM was added as induction. The growth curve was measured and shown in Figure.3.<br />
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  It could be found that for conditions of 4 or 6g/L maltose in M9 medium, when inducer arabinose was added as 1g/L, strains induced by 1mM tryptophan obtained much better growth after 12h than the corresponding negative control without tryptophan addition. In other conditions, this trend was not so significant,  which inferred that the condition for our sweet pressure part to work must be finely tuned. What is more, we have proved that tryptophan addition greatly  inhibited growth of cells (缺一个数据). Therefore,  the trend of better growth induced by tryptophan addition was very strong proof for the successful construction and function of our sweet pressure part, although it only worked in this particular condition.</p>
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<h2>[[File:4_3s.png‎|60px|left]] '''Part 3: THU-E Bitter Defender Part''' </h2>
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[[File:Plasmid3.png|446px|right]]
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<img width="446" src="/wiki/images/d/d9/Sweet.jpg" /> <br />
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   Figure.1 plasmid map for THU-E sweet pressure part </p>
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This is a plasmid providing selection pressure for the evolution and enrichment of  tryptophan overproduction microorganism phenotype by defending the toxicity of  tetracycline. This selection pressure was based on the tryptophan dependent tetracycline antiporterexpression which functioned in tetracycline culture condition.It is achieved by cloning<em> E. Coli</em> tetracycline  antiporter gene (<em>tetA</em>) downstream of our previously constructed tryptophan biosensor which wascontrolled by tac promoter in pTrc99A vector. Three different RBS (RBS-ori (RBS upstream of wild  type<em>E. Coli</em> tryptophanase(tnaA) operon), RBS-30 (B0030), RBS-32 (B0032)) were placed upstream of <em>tetA</em> gene.
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<p align="center"><img width="446" src="/wiki/images/d/da/Part4II.png"/><br />
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  Figure.2 Conception illustration of the working mechanism of sweet pressure part</p>
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Previously engineered <em>E. coli</em> with different tryptophan productivity (trp000, trp007, trp015 with the overall productivity as 0, 0.07, 0.15g/L, respectively. unpublished data) were used to test the performance  of this part. The tryptophan productivity of each strain wasmeasured, andit was clear that transformation of the engineered bitter defender parts didn’t significantly influence original  strains’ tryptophan relative productivity according to Figure.3.
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<p align="center"><img width="446" src="/wiki/images/0/04/Part3I.png" /><br />
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   Figure.3 Sweet part performance was measured by the dependent relation between the strain growth rate and the extracellular tryptophan addition in finely tuned M9 culture condition. Mal, ara and trp refer to maltose, arabinose and tryptophan, respectively. The units of them were g/L, g/L and mM, respectively.</p>
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We thus tested these three parts’ performance by measuring the growth rate of these nine strains (three kinds of strains with different tryptophan productivity carrying three kinds of selection pressure plasmids. They were named after trp000-30, trp000-32, trp000-ori, trp007-30, trp007-32, trp007-ori, trp015-30, trp015-32 and trp015-ori, respectively) in different tetracycline concentration. We interestingly  found that strains carrying bitter defender part with RBS B0030 and B0032 showed good tryptophan dependent growth property within the first 15h:
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(1) As the  increase of tetracycline, the selection pressure increased and the growth rate  of strains decreased.
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(2) With  increased selection pressure, it could be observed that within time window of  15h, in some selection pressure condition (tetracycline concentration) strains  with higher tryptophan productivity showed much higher growth rate, while the growth  of tryptophan non producer trp000 nearly inhibited by high concentration of  tetracycline.
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<p align="center"> <img width="446" src="/wiki/images/0/00/Part3II.png" /> <br />
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   Figure.1 Conception illustration of the working mechanism of bitter defender part <br /></p>
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<p align="center"> <img width="446" src="/wiki/images/5/59/Part4I.png" /> <br />
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Figure.2 Tryptophan productivity of  previously engineered strains with different productivity carrying three kinds of bitter defender plasmids<br /></p>
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<p align="center"> <img width="446" src="/wiki/images/c/cf/Part4Is.png" /> </p>
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<p align="center"><img width="446" src="/wiki/images/d/db/Part4Is2.png" /> <br />
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   Figure.3 Bitter defender part performance was measured by the dependent relation between the strain growth rate and the intracellular tryptophan productivity in finely tuned M9YE culture condition.Strains carrying bitter defender part with RBS B0030 and B0032 were shown respectively. </p>
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Latest revision as of 15:13, 27 September 2013

  • Totop

















4 3s.png
Part 3: THU-E Bitter Defender Part

Plasmid3.png

This is a plasmid providing selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype by defending the toxicity of tetracycline. This selection pressure was based on the tryptophan dependent tetracycline antiporterexpression which functioned in tetracycline culture condition.It is achieved by cloning E. Coli tetracycline antiporter gene (tetA) downstream of our previously constructed tryptophan biosensor which wascontrolled by tac promoter in pTrc99A vector. Three different RBS (RBS-ori (RBS upstream of wild typeE. Coli tryptophanase(tnaA) operon), RBS-30 (B0030), RBS-32 (B0032)) were placed upstream of tetA gene.

Previously engineered E. coli with different tryptophan productivity (trp000, trp007, trp015 with the overall productivity as 0, 0.07, 0.15g/L, respectively. unpublished data) were used to test the performance of this part. The tryptophan productivity of each strain wasmeasured, andit was clear that transformation of the engineered bitter defender parts didn’t significantly influence original strains’ tryptophan relative productivity according to Figure.3.

We thus tested these three parts’ performance by measuring the growth rate of these nine strains (three kinds of strains with different tryptophan productivity carrying three kinds of selection pressure plasmids. They were named after trp000-30, trp000-32, trp000-ori, trp007-30, trp007-32, trp007-ori, trp015-30, trp015-32 and trp015-ori, respectively) in different tetracycline concentration. We interestingly found that strains carrying bitter defender part with RBS B0030 and B0032 showed good tryptophan dependent growth property within the first 15h:

(1) As the increase of tetracycline, the selection pressure increased and the growth rate of strains decreased.

(2) With increased selection pressure, it could be observed that within time window of 15h, in some selection pressure condition (tetracycline concentration) strains with higher tryptophan productivity showed much higher growth rate, while the growth of tryptophan non producer trp000 nearly inhibited by high concentration of tetracycline.


Figure.1 Conception illustration of the working mechanism of bitter defender part


Figure.2 Tryptophan productivity of previously engineered strains with different productivity carrying three kinds of bitter defender plasmids


Figure.3 Bitter defender part performance was measured by the dependent relation between the strain growth rate and the intracellular tryptophan productivity in finely tuned M9YE culture condition.Strains carrying bitter defender part with RBS B0030 and B0032 were shown respectively.

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