Team:BOKU-Vienna/Experiments

Experiments

Overview

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"lucky shot" D.I.v.e.r.t.

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tRNA Priming

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FRT Terminaton strength

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self-splicing ribozyme

and the antibiotic resistance gene

Design

Selection of a self-splicing ribozyme:
For the proof of concept for D.I.V.E.R.T. an antibiotic resistance with an intron on the lagging strand is required. While in yeast there are plenty of well-described introns to choose from, in E. coli information is more rare. However, scientists already discovered group I and group II introns working in prokaryotes as well. For example, the Tetrahymena group I intron, or self-splicing ribozyme, is proven to exhibit splicing activity in vivo in E. coli without the expression of additional supporting proteins¹. In contrast to other self-splicing ribozymes the Tetrahymena group I intron can easily be engineered to cut itself scarlessly. For the exact recognition of the splicing sites intron-exon pairing sequences are present in the wild type mRNA. The most important of these regions are called P1 and P10 (paired region 1 and 10)². P1 determines the 5’ end of the self-splicing ribozyme and contains P1ex, which has a considerable influence on the splicing activity. P10 pairs with an exon sequence close to the 3’ end and is therefore signalling the 3’ end of the intron. Although P1 and P10 mark opposite ends of the intron, they are both located in the same region and it is worth noticing that P1 and P10 can overlap.

Figure 1: Design of the precursor mRNA. Exon bases are written in lowercase, the Tetrahymena group I intron bases in capital letters. A part of the P1 region is complementary to the 5’ exon sequence determining the 5’ end of the intron, whereas the P10 region is determining the 3’ end.

Selection of an antibiotic resistance:
For the Tetrahymena group I intron Guo et al. proposed in their publication³ that the 5’ adjacent exon sequence of the mRNA should consist of CUCUCU. Concerning this and the requirement of the intron being on the lagging strand we were looking for an antibiotic resistance with AGAGAG (reverse complementary to CUCUCU). We finally chose an ampicillin resistance, a β-lactamase5, which exhibits AGAGAA approximately in the middle of the gene⁴. By introducing a silent mutation (A357G) the desired 5’ adjacent exon sequence could be obtained. Furthermore, a BsaI recognition site (T718, C719, T720) was silently mutated to comply with our Golden Gate-cloning standard.

Design of the self-splicing ribozyme:
When the Tetrahymena group I intron is inserted into a foreign gene, the adjustment of P1 and P10 are crucial for the preservation of the self-splicing ability: The P10 region was exchanged for bases complementary to the 3’ adjacent exon sequence of the new β-lactamase gene. Because P1ex and P10 are overlapping, P1 was altered as well. Additionally, as Guo et al. suggests³, a non-complementary base pair was introduced in the P1ex sequence to promote splicing activity.

Figure 2: Design of the precursor mRNA of the β-lactamase gene with the engineered Tetrahymena intron . Exon bases are written in lowercase, intron bases in capital letters. The β-lactamase sequence is reverse complementary due to the proof of concept setup. P1 as well as P10 were altered in the process of the design.

Experiments & Results

Proof of self-splicing:
To test the functionality of the engineered self-splicing Tetrahymena group I intron with the adjacent β-lactamase exon sequence, it was cloned into an expression vector and transformed into E. coli. The total mRNA was then purified and and a cDNA synthesis was performed using gene specific primer. For enhancing the amount of detectable DNA a PCR with was done afterwards. A gel electrophoresis was performed where both, spliced, and unspliced DNA was visible.

Figure 3: The sample was loaded on a agarose gel. After 40 min at 130 V two bands were visible. One 1248 bp long represents the unspliced mRNA, the second, 835 bp long, represents the spliced mRNA.

The 835 bp band was cut out and cleaned up. By performing a PCR the DNA was multiplied and subsequently sent for sequencing, which confirmed the self-splicing activity exactly as it was intended during the design.

Ampicillin resistance:
For the proof of concept a functional ampicillin resistance gene is required and, since the recombination is Flp/FRT mediated, a FRT sequence is introduced after the start and stop codon. Therefore, after Met 16 additional amino acids are expressed at the N-terminus of the ampicillin resistance gene. To test if the ampicillin resistance is still functional, this modified gene is cloned into an expression vector. After subsequent transformation into chemical competent E. Coli DH10b, cells are plated on LB low salt agar with 100 µg/mL ampicillin concentration. After an overnight incubation ampicillin resistant colonies were visible. For verification a colony was picked and its plasmid was purified and the instert sequenced.

[1]: Waring RB, Ray JA, Edwards SW, Scazzocchio C, Davies RW. The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function. Cell. 1985 Feb;40(2):371-80.

[2]: Burke JM, Cech TR, Davies RW, Schweyen RJ, Shub DA, Szostak JW, Tabak HF. Structural conventions for group I introns. Nucleic Acids Res. 1987 Sep 25; 15(18): 7217–7221.

[3]: Guo F, Cech TR. In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron. RNA. 2002 May;8(5):647-58.

[4]: Zhou W, Wang Y, Lin J. Functional cloning and characterization of antibiotic resistance genes from the chicken gut microbiome.Appl Environ Microbiol. 2012 Apr;78(8):3028-32. doi: 10.1128/AEM.06920-11. Epub 2012 Jan 27.

CRISPR assisted integration

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