Difference between revisions of "Part:BBa K2443038"
Line 3: Line 3:
 
<partinfo>BBa_K2443038 short</partinfo>
 
<partinfo>BBa_K2443038 short</partinfo>
  
−
tRNA<sup>Phe</sup>-MS2 co-purification construct.
+
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>
  
−
<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 "http://2017.igem.org/Team:Lethbridge/Design#anchor5" tRNA purification</a> section here.</p>
 
  
−
<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>
+
<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 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>
  
−
"https://static.igem.org/mediawiki/2017/b/b8/Lethbridge_trnapurification1.png"
+
<p>  For more information on the design,  see the "http://2017.igem.org/Team:Lethbridge/Design#anchor5" tRNA purification</a> section here.</p>
 +
 
 +
https://static.igem.org/mediawiki/2017/9/92/Lethbridge_tRNApurification.png
  
 
<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).
 
<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).
Line 18: Line 19:
  
  
−
"https://static.igem.org/mediawiki/2017/9/92/Lethbridge_tRNApurification.png"
+
https://static.igem.org/mediawiki/2017/9/92/Lethbridge_tRNApurification.png
  
 
<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).  
 
<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).  

Revision as of 01:49, 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 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 tRNAPhe, as shown in Figure 4.

For more information on the design, see the "http://2017.igem.org/Team:Lethbridge/Design#anchor5" tRNA purification</a> section here.

Lethbridge_tRNApurification.png

Figure 3 - 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).


Lethbridge_tRNApurification.png

Figure 4 - 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).


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]