Next vivo was designed in such a way that we could test multiple components and modules individually while developing the full system. These include:
- Evaluating the efficacy of multi-protein purification using TX-TL proteins
- Developing a universal tool to characterize TX-TL systems
- Designing and testing a novel tRNA purification strategy
- Testing the TX components of our system for activity
One of the key features of Next vivo is the ability to purify all of the components in a single step purification. As a proof of concept we expressed four of the TX-TL components and co-purified them all using a Nickel Sepharose chromatography column (Figure 1). The four proteins used in this initial test were selected based on their molecular weights relative to each other for visualization purposes.
Figure 1 - Representative overexpression and multi-protein purification of TX-TL components. Each TX-TL component was expressed from E. coli cells carrying the plasmid encoding the specified component and samples three hours post induction were collected. The expressing cells of each component were pooled and lysed before applying the lysate to a Nickel Sepharose affinity column for isolation of just the hexahistidine tagged TX-TL components. After washing away the unwanted cellular proteins and debris, the TX-TL components were eluted from the Nickel Sepharose to a high level of purity. Lanes are as follows: 1- Protein ladder; 2- HisRS overexpression; 3- TrpRS overexpression; 4- RF3 overexpression; 5- RRF overexpression; 6- HisRS, TrpRS, RF3 and RRF elution. Protein Sizes: HisRS 48.3kDa; TrpRS 38.8kDa; RF3 60.9kDa; RRF 21.9 kDa
From this initial test we have confidence in the feasibility of scaling up the multi-protein purification to include all, or large groups of, the TX-TL proteins. The overexpression and purification of these four proteins was done with minimal lab equipment and supplies.
We successfully co-purified 4 of our translation proteins!
The biggest issue we initially faced in developing Next vivo was determining how we could purify tRNA quickly and efficiently. The solution we decided upon was an adapted MS2 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 tRNA purification section here.
Both the tRNAPhe-MS2 construct and MS2BP were expressed individually in E. coli BL21-Gold (DE3) cells. Upon which time the cells were lysed, the lysates combined, and applied to a Nickel Sepharose affinity column. The MS2BP is able to bridge the Nickel Sepharose column and the tRNA-MS2 allowing the tRNA to be isolated from the cell lysate.
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).
We successfully purified tRNAPhe using our novel purification technique!
As an additional tool outside the Next vivo system, we developed a construct that can be used to measure the amount of transcription, translation, or both! The details of this construct can be found on the design page. Using a purified T7 polymerase, we in vitro transcribed the full validation construct. After adding DHFBI, the fluorophore, we were able to observe green fluorescence (Figure 3).
Figure 3 - Characterization of the EYFP-Spinach validation construct. A In vitro transcribed Spinach RNA visualized on 1% agarose for 30 min at 100 V. Lane 1- 1kb ladder and Lane 2- Spinach RNA (approximately 900 nt). B Fluorimeter data illustrating green fluorescence following addition of DFHBI, with an excitation of 447nm and emission of 497nm.
From these results we are confident that we can produce the Spinach RNA and use it as a measure of transcriptional activity for our Next vivo system or T7 polymerase alone.
We successfully transcribed Spinach RNA!
Work on the Next vivo system will not end with the iGEM competition and will be carried on by several team members going forward (see Next vivo 2.0).
- Perform the multi-protein purification with all Next vivo TX-TL components
- Test purified tRNAPhe for aminoacylation efficiency (how efficient the amino acid can be attached)
- Design and order the remaining 19 tRNA needed to encode the 20 standard amino acids
- Insert the MS2 tag into the ribosomal RNA genes to test purification of the ribosome
- Test Next vivo purified release factors (translation components) in the PURExpress kits lacking these factors for viability
- Design a troubleshooting module to determine which components are non-functional