Team:TU Dresden/Demonstrate
Beaming BioBricks from Space...
Demonstrate
“Simple can be harder than complex: You have to work hard to get your thinking clean to make it simple. But it’s worth it in the end because once you get there, you can move mountains.“
Steve Jobs
The most complicated engineering is often required to develop the most simple method.
As a multi-component project that depended on a lot on separate parts to work independently, DipGene was subjected to a lot of independent “Killer-experiments” that could have each in itself disproven the basic concept of our project idea.
In the following you will find all the separate components that had to be proven to work and the experiments that we performed to investigate them.
We developed a new method based on immobilizing DNA on cellulose paper. We optimized buffers and incubation times to get ideal DNA quality and amounts. The idea is based on a publication by Zou et al. 2017.
This method was used to extract DNA from E.coli (1.5 mL overnight cultures)
Figure 1 – The extraction of DNA from microbial cells
This method was used by the Spirulina team for all of their PCRs.
Figure 2 – The extraction of DNA from cyanobacteria cells
This method was used to extract genomic DNA from human epithelial cells and amplify the region of interest in PCR.
Figure 3 – The extraction of DNA from human cells
The protocols for all of these methods can be found on our Protocols page
To follow the cloning procedure of our full construct please refer to the notebook, where we also characterized different expression and purification conditions.
This method was used to extract DNA from E.coli (1.5 mL overnight cultures)
Figure 4 – The final construct made up of five different BioBricks, translationally fused by using RCF25, introduced into the expression plasmid (K3037000) by using RCF10; MBP - Maltose Binding Protein, Linker, HRP - Horseradish Peroxidase, dCas9* - dead Cas9 enzyme with a mutation to exclude forbidden EcoRI site and a Strep - Tag; newly designed BioBricks from Team TU Dresden in bold, old BioBricks used from other Teams in normal font
The full construct consisted of five different BioBricks that were assembled in a sequential manner (Figure 4). The first ever expression of this protein was followed over seven hours, samples were taken every hour and loaded onto an SDS-PAGE (Figure 5).
Figure 5 – Expression of full construct in pOCC97 not optimized (Bba_K3037000)
In the picture the successful expression of the full construct can be seen. It is a novel fusion protein of 230 kDa size. The full construct is uploaded in K3037005. For expression it was cloned in K3037000 and grown overnight at 37 °C with 1mM IPTG. The band can be observed at the expected size, between the marker lanes for 184 and 245 kDa. After the first hour of induction a continuous increase of our protein can be seen, while the native proteins (background of the cell lysate stay constant).
For a fusion protein,consisting of so many different parts, it is important to show that each single part works as expected. Since it could be possible that they influence each other in folding and function.
1. Prove that MBP is intact via amylose resin purification
Figure 6 – SDS-PAGE showing that MBP specifically binds to the resin
The protein of interest was successfully purified via batch-binding on amylose resin. This shows that the MBP-tag is intact and able to specifically bind to the resin (Figure 6). Many truncated version of our protein of interest can be seen, this typically happens for very large proteins in E.coli, because the bacteria often interrupts expression.
Figure 7 – Full Construct containing functional dCas9
The loading scheme was as indicated in the image.
The sry gene was amplified by PCR, to have high DNA amounts.
The concentration of the Full Construct was varied from 0.64 to 4.56 ug to determine the minimal concentration at which a shift was still observable.
Figure 7 shows that the minimal concentration at which our expressed and purified Full Construct is at 1.28 ug.
Investigating the activity of HRP in the full construct was done in a dynamic assay. The absorbance at 650 nm was measured over time with the substrate TMB which is colorless and converted into a blue product by oxidation through HRP.
Figure 8 – Activity assay full construct compared to HRP
Video 1 – Proof that HRP, as part of the full construct, is able to oxidize TMB to colorful product (color shift to blue). Reaction product stabilizes at low pH (color shift to yellow).
One of the basic ideas of our project was relying on the interaction between dCas9 and its target DNA. This reaction is well characterized in solution, but has never been shown to be working on a matrix substrate such as a cellulose disk. Many aspects influence the binding ability of this protein and therefore it was of great importance to investigate if the immobilization of DNA on the cellulose would influence this interaction.
To prove that DNA binding was not impaired, DNA was immobilized on a cellulose disk, then incubated with a dCas9-guide RNA mixture for one hour at 37 °C and the cellulose was loaded directly into the well of an EMSA shift essay . A cellulose disk with the target DNA was loaded in the lane next to it, without dCas9-guide RNA to prove that the DNA could leave the cellulose in the essay (Figure 9)
Figure 9 – dCas9 can still bind to the DNA immobilized on the cellulose paper
Loading scheme was:
Lane 11 - sry + dCas9-GFP+Guide RNA (1,4,2)
Lane 12 - sry gene loaded on cellulose strip
The last lanes (11,12) are investigating this issue:
The sry gene was amplified by PCR, to have high DNA amounts. The DNA was immobilized on the cellulose and then incubated with a dCas9-guide RNA mixture for 1 hour at 37 °C. This way dCas9 could only bind to the target DNA and cause a shift in the EMSA, if the cellulose matrix would not impair its binding ability. In the last lane, lane 12, the sry gene alone was loaded on the cellulose to prove that it was able to leave the cellulose matrix.
A faint band can be seen in lane 11 at the same height of the shift that can be observed in lanes 2 and 4-6. (loaded with the same reaction in solution) This proves that the binding to DNA is still possible on the cellulose matrix. It seems to be slightly impaired compared to the solution reaction, since the band is fainter. But proves that the basic idea that our project is relying on is possible.
Figure 10 – EMSA showing that the eGFP-dCas9 does not bind nonspecifically to the cellulose disks and can be washed away with the W1 buffer.
The loading scheme was as indicated following:
Lane 1 - Marker
Lane 2 - sry
Lane 3 - dCas9-HRP + (1,4,2 guide RNA) + sry
Lane 4 - dCas9 + gRNA (1,4 and 2) first incubated and then washed once and then sry gene.
Lane 5 - dCas9 + gRNA (1,4 and 2) first incubated and then washed twice and then sry gene.
The sry gene was amplified by PCR, to have high DNA amounts.
A critical part of our project design was to develop an easy method to selectively immobilize DNA on a cellulose matrix, and tailor buffers that can be used to wash away proteins and debris that could interfere with our dCas9-HRP fusion protein in binding to the target DNA region. We optimized the wash buffer composition to selectively wash proteins out of our cellulose matrix, while leaving nucleic acids immobilized in the disks.
In lane 4 and 5 of Figure 10 we do not see any pull of the sry gene indicating that all the unbound Full Construct (dCas9) was removed during the washing step. This suggests that the dCas9 only binds to the cellulose in the presence of target DNA.
Hence, Figure 10 proves the specificity of our Full Construct (dCas9) protein interaction to the cellulose and it functions only in the presence of specific guideRNAs and target DNA.
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