Latest revision as of 02:06, 2 November 2017

tRNAPhe-MS2

tRNA^Phe-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 tRNA^Phe, 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 tRNA^Phe 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 tRNA^Phe 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