Difference between revisions of "Part:BBa K2443038"
 
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<partinfo>BBa_K2443038 short</partinfo>
 
<partinfo>BBa_K2443038 short</partinfo>
  
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tRNA<sup>Phe</sup>-MS2 co-purification construct.
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tRNA<sup>Phe</sup>-MS2 co-purification construct. Contains a T7 promoter -10 followed by tRNA Phe, two MS2 RNA aptamers and a double terminator. To be used in conjunction with BBa_K2109108.
  
  
 
<h1>tRNA Purification</h1>
 
<h1>tRNA Purification</h1>
  
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<p>The biggest issue we initially faced in developing Next <i>vivo</i> was determining how we could purify tRNA quickly and efficiently. The solution we decided upon was an adapted MS2 RNA co-purification combined with a subsequent incubation with RNase H and a DNA oligo that would selectively cleave and release a tRNA of the proper size. For more information on the design,  see the <a href="http://2017.igem.org/Team:Lethbridge/Design#anchor5" id="pageLink"> tRNA purification</a> section here.</p>
 
  
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<p>Both the tRNA<sup>phe</sup>-MS2 aptamer construct and MS2BP were overexpressed individually in <i>E. coli</i> BL21-Gold (DE3) cells. Upon which time the cells were lysed, the lysate combined, and applied to a Nickel Sepharose affinity column. In order to cleave the RNA, 1 µM of DNA oligo was added to the column, as well as varying amounts of RNase H. Incubation times on the column with RNase H and DNA oligo varied from 2 hours (Figure 3) to 12 hours (Figure 4), and the amount of RNase H used varied from 10 units to 100 units (Figure 3 and 4). Based upon the varied conditions, a longer incubation time had the greatest effect on tRNA cleavage efficiency with units of RNase H being optimal between the range of 5-50. With these improvements from our initial attempt at tRNA cleavage we successfully purified tRNA<sup>Phe</sup>, as shown in Figure 4.</p>
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<p>Both BBa K2443038 and BBa_K2109108 were overexpressed individually in <i>E. coli</i> BL21-Gold (DE3) cells. Upon which time the cells were lysed, the lysate combined, and applied to a Nickel Sepharose affinity column. In order to cleave the RNA, 1 µM of DNA oligo was added to the column, as well as varying amounts of RNase H. Incubation times on the column with RNase H and DNA oligo varied from 2 hours (Figure 1) to 12 hours (Figure 2), and the amount of RNase H used varied from 10 units to 100 units (Figure 1 and 2). Based upon the varied conditions, a longer incubation time had the greatest effect on tRNA cleavage efficiency with units of RNase H being optimal between the range of 5-50. With these improvements from our initial attempt at tRNA cleavage we successfully purified tRNA<sup>Phe</sup>, as shown in Figure 2.</p>
  
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"https://static.igem.org/mediawiki/2017/b/b8/Lethbridge_trnapurification1.png"
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<p>  For more information on the design, see the tRNA purification section of our wiki ("http://2017.igem.org/Team:Lethbridge/Design#anchor5")</p>
  
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<p><b>Figure 3 - Preliminary tRNA<sup>Phe</sup> Purification. </b>12% 8M urea PAGE run for 45 mins at 300 V. All concentrated fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH<sub>2</sub>O. Lanes are as follows: 1- tRNA fraction with 20 units of RNase H added; 2- concentrated tRNA fraction 20 units of RNase H added; 3- concentrated MS2 fraction 1 20 units of RNase H added; 4- concentrated MS2 fraction 2  20 units of RNase H added; 5- tRNA standard (76 nt).
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<p><b>Figure 1 - Preliminary tRNA<sup>Phe</sup> Purification. </b>12% 8M urea PAGE run for 45 mins at 300 V. All concentrated fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH<sub>2</sub>O. Lanes are as follows: 1- tRNA fraction with 20 units of RNase H added; 2- concentrated tRNA fraction 20 units of RNase H added; 3- concentrated MS2 fraction 1 20 units of RNase H added; 4- concentrated MS2 fraction 2  20 units of RNase H added; 5- tRNA standard (76 nt).
 
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<img src="https://static.igem.org/mediawiki/2017/9/92/Lethbridge_tRNApurification.png" width=50% height=50% />
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"https://static.igem.org/mediawiki/2017/9/92/Lethbridge_tRNApurification.png"
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<p><b>Figure 2 - Successful tRNA<sup>Phe</sup> Purification. </b>12% 8M urea PAGE run for 45 mins at 300 V. All fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH<sub>2</sub>O. Lanes are as follows - 1- MS2 fraction 25 units of RNase H added; 2- tRNA fraction 25 units of RNase H added; 3- MS2 fraction 50 units of RNase H added; 4- tRNA fraction 50 units of RNase H added; 5- MS2 fraction 100 units of RNase H added; 6- tRNA fraction 100 units of RNase H added; 7- MS2 fraction 10 units of RNase H added; 8- tRNA elution 10 units of RNase H added; 9- tRNA standard (76 nt).  
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<p><b>Figure 4 - Successful tRNA<sup>Phe</sup> Purification. </b>12% 8M urea PAGE run for 45 mins at 300 V. All fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH<sub>2</sub>O. Lanes are as follows - 1- MS2 fraction 25 units of RNase H added; 2- tRNA fraction 25 units of RNase H added; 3- MS2 fraction 50 units of RNase H added; 4- tRNA fraction 50 units of RNase H added; 5- MS2 fraction 100 units of RNase H added; 6- tRNA fraction 100 units of RNase H added; 7- MS2 fraction 10 units of RNase H added; 8- tRNA elution 10 units of RNase H added; 9- tRNA standard (76 nt).  
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<p> Additional optimization of cleavage conditions and wash steps will be needed in order to optimize this process.</p>
  
  

Latest revision as of 02:06, 2 November 2017


tRNAPhe-MS2

tRNAPhe-MS2 co-purification construct. Contains a T7 promoter -10 followed by tRNA Phe, two MS2 RNA aptamers and a double terminator. To be used in conjunction with BBa_K2109108.


tRNA Purification


Both BBa K2443038 and BBa_K2109108 were overexpressed individually in E. coli BL21-Gold (DE3) cells. Upon which time the cells were lysed, the lysate combined, and applied to a Nickel Sepharose affinity column. In order to cleave the RNA, 1 µM of DNA oligo was added to the column, as well as varying amounts of RNase H. Incubation times on the column with RNase H and DNA oligo varied from 2 hours (Figure 1) to 12 hours (Figure 2), and the amount of RNase H used varied from 10 units to 100 units (Figure 1 and 2). Based upon the varied conditions, a longer incubation time had the greatest effect on tRNA cleavage efficiency with units of RNase H being optimal between the range of 5-50. With these improvements from our initial attempt at tRNA cleavage we successfully purified tRNAPhe, as shown in Figure 2.

For more information on the design, see the tRNA purification section of our wiki ("http://2017.igem.org/Team:Lethbridge/Design#anchor5")

Figure 1 - Preliminary tRNAPhe Purification. 12% 8M urea PAGE run for 45 mins at 300 V. All concentrated fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH2O. Lanes are as follows: 1- tRNA fraction with 20 units of RNase H added; 2- concentrated tRNA fraction 20 units of RNase H added; 3- concentrated MS2 fraction 1 20 units of RNase H added; 4- concentrated MS2 fraction 2 20 units of RNase H added; 5- tRNA standard (76 nt).

Figure 2 - Successful tRNAPhe Purification. 12% 8M urea PAGE run for 45 mins at 300 V. All fractions were phenol chloroform extracted, ethanol precipitated and re-suspended in 30 µL of ddH2O. Lanes are as follows - 1- MS2 fraction 25 units of RNase H added; 2- tRNA fraction 25 units of RNase H added; 3- MS2 fraction 50 units of RNase H added; 4- tRNA fraction 50 units of RNase H added; 5- MS2 fraction 100 units of RNase H added; 6- tRNA fraction 100 units of RNase H added; 7- MS2 fraction 10 units of RNase H added; 8- tRNA elution 10 units of RNase H added; 9- tRNA standard (76 nt).

Additional optimization of cleavage conditions and wash steps will be needed in order to optimize this process.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]