Difference between revisions of "Part:BBa K1781000"

Latest revision as of 03:53, 2 November 2017

P3 from M13

M13 gene 3 coding for infection protein P3

[http://2017.igem.org/Team:Heidelberg Team Heidelberg 2017] was further characterized by introducing it into a new RFC (http://2017.igem.org/Team:Heidelberg/RFC), as well as making it broadly available in our Phage-assisted continous evolution (PACE) and Phage-related discontinous evolution (PREDCEL) toolbox (http://2017.igem.org/Team:Heidelberg/Toolbox). (see: BBa_K2398006)
(--moritz_p95 4:45, 02.11.2017 (UTC))

This part provide bacteriophage M13 geneIII with a weak RBS, sd8[1], flanked by two homology regions for the usage due to the cloning standard of the iGEM Team Heidelberg 2017 (http://2017.igem.org/Team:Heidelberg/RFC). Figure one gives a short overview of our standard. Our BioBricks from the registry can easily be used for the assembly of blasmid with the standard (Fig.: 2).

Figure 1: In our cloning standard, compatible building blocks are defined by specific functionalities. They are flanked by defined homology regions, indicated by numbers, which are necessary for the assembly of the APs with the Gibson method. This results in a highly customizable plasmid, composed of the desired origin of replication, an antibiotic resistance (4-5), a bicistronic operon with geneIII (2-3)and the desired reporter (3-4), which can be activated by any promoter (1-2)and a second expression cassette for additional genes that are necessary for the respective circuit (1-5).

Figure 2: Compatibility of our cloning stadard with the RFC10;Any AP building block can be cloned into RFC[10] standard by inserting BglII sites between the homology regions and the biobrick prefix or suffix, respectively. To use such a part for AP assembly, it has to be digested with BglII. The resulting fragment should be purified and can subsequently used for Gibson assembly with other parts.

Phage-assisted continuous evolution (PACE) is a powerful in vivo directed evolution method invented by Kevin Esvelt (now at the MIT media lab) and David Liu (Harvard University) (Esvelt et al, Nature, 2011). It can in principle be applied for directed evolution of any gene or protein of interest (GOI and POI, respectively), provided that the function to be evolved can be coupled to gene expression in E. coli (Esvelt et al, Nature, 2011). Conceptually, PACE mimics a closed evolution circle composed of (i) variant replication, (ii) mutation and (iii) selection in a bioreactor. The basic principle of PACE is shown in Figure 1. In brief, PACE employs M13 phages that encode protein of interest (POI) to be evolved. These phages have a knockout for gene III, an essential M13 gene essential for phage replication. }}

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Figure 3: Basic principle of phage-assisted, continuous evolution. (see main text for details).

During PACE, the POI-encoding phages are propagated of E. coli host cells carrying two plasmids. One it the mutagenesis plasmid (MP), which encodes highly mutagenic genes strongly reducing phage replication fidelity and error repair. As these genes are toxic to the E. coli host, they are set under control of an arabinose-inducible pBAD promoter. Different MP variants have been reported, which cause mutation rates up to ~2.3 substitutions per kb during phage replication when fully induced (Badran et al, Nature Communications, 2015), thereby highly accelerating the evolution.

The second plasmid is called accessory plasmid (AP), and is the PACE component inducing the selection pressure directing the evolution towards the desired goal. The AP thereby encodes gene III, the aforementioned essential M13 gene required for phage replication. Importantly, gene III expression from the AP is made dependent on the function of the phage-encoded POI, e.g. via a synthetic circuit. In other words: the AP induces a selection pressure towards optimizing POI for the function required to activate gene III expression. In the simple case of evolving a transcription factor as depicted in Figure 1, gene III would simply be expressed from the promoter (“pTarget”) the transcription factor should be optimized for.

Results – evolving split T7 polymerase toward improved auto-reassembly

We set out to employ PACE for evolving a split T7 polymerase towards increased auto-reassembly. Details on the how we obtained the particular split variant used in this PACE experiment can be found on our (http://2017.igem.org/Team:Heidelberg/Protein_Interaction). In brief, based on the paper by Tiun Han et al. (ACS Synthetic Biology, 2017), we tested M13 phages encoding 4 different split T7 variants for their ability to propagate on an E. coli selection strain transformed with a pT7-geneIII accessory plasmid. The variant encoding the split site at T7 residues 564/565 showed highest propagation and was therefore chosen for further optimization via PACE. We generated a PACE strain, by co-transforming our T7-gene III AP and MP4 (Badran et al, Nature Communications, 2015). Then, by using our custom-build setup, we performed 30 hours of in vivo directed evolution via PACE.

Phages are continuously propagated on a mutagenic E. coli selection strain, carrying an AP comprising of a T7 promoter driving gene III. Thereby, variants should evolve, which show improved split T7 function.

During the PACE run continuously monitored the phage titer as well as optical density of our E. coli hosts (Figure 4). The used flow rate was approximately 1 lagoon volume per hour and adapted according to the optical density measurements.

(A) Phage titer was estimated by manual sampling every 5 hours followed by plague essay. (B) Samples were taken every two hours and OD600 was determined using a nanophotometer. During the whole PACE run, the optical density is located in the range of 0.5 to 1, which is equivalent to the reaction conditions reported in the literature (Esvelt et al, Nature, 2011)

After having finished our PACE run, we performed plague assays and PCR amplified the Split T7 genes from 5 different plagues followed by sanger-sequenced. As hoped, we observed a recurrent, coding mutation (T877P) present in three out of the five sequenced split T7 variants (Figure 5).

Following three days of in vivo evolution with PACE, a plague assay was performed and the split T7 insert of five individual phage clones was analyzed by sanger-sequencing. We observed a recurrent mutation (T877P) in three out of the five clones, suggesting an evolutionary advantage (i.e. increased fitness) of the corresponding split T7 mutant as compared its non-mutated counterpart.

Interestingly this residue is located very close to the interaction surface of the two split T7 domains, suggesting a possible role in T7 auto-reassembly (Figure 6).

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References

[1] Ringquist, S.; Shinedling, S.; Barrick, D.; Green, L.; Binkley, J.; Stormo, G. D.; Gold, L. (1992): Translation initiation in Escherichia coli: sequences within the ribosome-binding site. In: Molecular microbiology 6 (9), S. 1219–1229.

Sequence and Features

Functional Parameters

Some difficulties were found with the expression of P3 and the resulting washout of the phages. The plasmid showed itself stable during the whole cultivation which makes the next step a cultivation without antibiotics possible and highly desirable.

Further experiments regarding the plasmid stability should be performed, as well as reducing the yeast extract from the medium by identifying the missing element in the minimal medium. A special expression analysis has to be done on P3 in relation to the IPTG-concentration.

In order to ensure proper antibiotic denaturation the cultivated medium had to be sterilized for 20 minutes at 121 °C. Further experiments are necessary to test the need of antibiotics for plasmid stability in the system. Since the antibiotic resistance of the recombinant E. coli strand is based on chloramphenicol degradation..

Additionally, the synthesized enzyme is secreted into the cultivation medium. This might lead to a complete loss of antibiotic function and therefore allow plasmid free E. coli to reproduce. As a result, the plasmid free cells might accumulate inside the CSR. Thus it is necessary to compare the plasmid stability of antibiotic free and antibiotic enriched media. Accounting to the previous reasons the influence of the chloramphenicol on plasmid stability might be negligible. As a result other ways to support plasmid stability or antibiotic free systems might be used for further experiments.