The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as non-coding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU, but the remaining thirteen "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multi-dimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To further evaluate synonymous and non-synonymous alternatives to essential AGRs, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild type allele, allowing us to exhaustively evaluate the fitness impact of all 64 codon alternatives. Using this method, we confirmed relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.
Significance Statement This work presents the genome-wide replacement of all rare AGR arginine codons in the essential genes of Escherichia coli with synonymous CGN alternatives. Synonymous codon substitutions can lethally impact non-coding function by disrupting mRNA secondary structure and ribosomal binding site-like motifs. Here we quantitatively define the range of tolerable deviation in these metrics and use this relationship to provide critical insight into codon choice in recoded genomes. This work demonstrates that genome-wide removal of AGR is likely to be possible, and provides a framework for designing genomes with radically altered genetic codes.
The genetic code possesses inherent redundancy (1), with up to six different codons specifying a single amino acid. While it is tempting to approximate synonymous codons as equivalent (2), most prokaryotes and many eukaryotes (3, 4) display a strong preference for certain codons over synonymous alternatives (5, 6). While different species have evolved to prefer different codons, codon bias is largely consistent within each species (5). However, within a given genome, codon bias differs among individual genes according to codon position, suggesting that codon choice has functional consequences. For example, rare codons are enriched at the beginning of essential genes (7, 8), and codon usage strongly affects protein levels (9-11), especially at the N-terminus (12). This suggests that codon usage plays a poorly understood role in regulating protein expression. Several hypotheses attempt to explain how codon usage mediates this effect, including but not limited to: facilitating ribosomal pausing early in translation to optimize protein folding (13); adjusting mRNA secondary structure to optimize translation initiation or to modulate mRNA degradation; preventing ribosome stalling by co-evolving with tRNA levels (6); providing a "translational ramp" for proper ribosome spacing and effective translation (14); and providing a layer of translational regulation for independent control of each gene in an operon (15). Additionally, codon usage may impact translational fidelity (16), and the proteome may be tuned by fine control of the decoding tRNA pools (17). Although Quax et al. provide an excellent review of how biology chooses codons, systematic and exhaustive studies of codon choice in whole genomes are lacking (18). Studies have only begun to empirically probe the effects of codon choice in a relatively small number of reporter genes (12, 19-22). Several important questions must be answered as a first step towards designing custom genomes exhibiting new functions—How flexible is genome-wide codon choice? How does codon choice interact with the maintenance of cellular homeostasis? What heuristics can be used to predict which codons will conserve genome function?
Replacing all essential instances of a codon in a single strain would provide valuable insight into the constraints that determine codon choice and aid in the design of recoded genomes. Although the UAG stop codon has been completely removed from Escherichia coli (23), no genome-wide replacement of a sense codon has been reported. While the translation function of the AGG codon has been shown to permit efficient suppression with nonstandard amino acids (24-26), AGG necessarily remains translated as Arg in each of these studies. No study has yet demonstrated that all AGR codons (or all instances of any sense codon) can be removed from essential genes, nor explained why certain AGR codons could not be changed successfully. These insights are crucial for unambiguously reassigning AGR translation function.
We chose to study the rare arginine codons AGA and AGG (termed AGR according to IUPAC conventions) because the literature suggests that they are among the most difficult codons to replace and that their similarity to ribosome binding sequences underlies important non-coding functions (8, 27-30). Furthermore, their sparse usage (123 instances in the essential genes of E. coli MG1655 and 4228 instances in the entire genome (Table 1, S1)) made replacing all AGR instances in essential genes a tractable goal, with essential genes serving as a stringent test set for identifying any fitness impact from codon replacement (31). Additionally, recent work has shown the difficulty of directly mutating some AGR codons to other synonymous codons (25), although the authors do not explain the mechanism of failure or report successful implementation of alternative designs. We attempted to remove all 123 instances of AGR codons from essential genes by replacing them with the synonymous CGU codon. CGU was chosen to maximally disrupt the primary nucleic acid sequence (AGR→CGU). We hypothesized that this strategy would maximize design flaws, thereby revealing rules for designing genomes with reassigned genetic codes. Importantly, individual codon targets were not inspected a priori in order to ensure an unbiased empirical search for design flaws.
Summary of AGR codons changed by location in the genome, and failure rates by pool.
To construct this modified genome, we used co-selection multiplex automatable genome engineering (CoS-MAGE) (32, 33) to create an E. coli strain (C123) with all 123 AGR codons removed from its essential genes (Figure 1A and see Table S1 for a complete list of AGR codons in essential genes). CoS-MAGE leverages lambda red-mediated recombination (34, 35) and exploits the linkage between a mutation in a selectable allele (e.g. tolC) to nearby edits of interest (e.g., AGR conversions), thereby enriching for cells with those edits (Figure S1). To streamline C123 construction, we chose to start with E. coli strain EcM2.1, which was previously optimized for efficient lambda red-mediated genome engineering (33, 36). Using CoS-MAGE on EcM2.1 improves allele replacement frequency by 10-fold over MAGE in non-optimized strains but performs optimally when all edits are on the same replichore and within 500 kilobases of the selectable allele (33). To accommodate this requirement, we divided the genome into 12 segments containing all 123 AGR codons in essential genes. A tolC cassette was moved around the genome to enable CoS-MAGE in each segment, allowing us to rapidly prototype each set of AGR→CGU mutations across large cell populations in vivo. (Please see the 'General Replacement Strategy' and 'Troubleshooting Strategy' sections of the Materials & Methods for a more detailed discussion). Of the 123 AGR codons in essential genes, 110 could be changed to CGU by this process (Figure 1), revealing considerable flexibility of codon usage for most essential genes. Allele replacement (in this case, AGR→CGU codon substitution) frequency varied widely across these 110 permissive codons, with no clear correlation between allele replacement frequency and normalized position of the AGR codon in a gene (Figure 2A).
Workflow used to create and analyze strain C123. The DESIGN phase involved identification of 123 AGR codons in the essential genes of Escherichia coli. MAGE oligos were designed to replace all instances of these AGR codons with the synonymous CGU codon. The BUILD phase used CoS-MAGE to convert 110 AGR codon to CGU and to identify 13 AGR codons that required additional troubleshooting. The in vivo TROUBLESHOOTING phase resolved the 13 codons that could not be readily converted to CGU and identified mechanisms potentially explaining why AGR→CGU was not successful. In the STUDY Phase, next-generation sequencing, evolution and phenotyping was performed on strain C123. (outer) Schematic of the C123 genome (Nucleotide 0 oriented up; numbering according to strain MG1655). Exterior labels indicate the set groupings of AGR codons. Successful AGR→CGU (110 instances) conversions are indicated by radial green lines, and recalcitrant AGR codons (13 instances) are indicated by radial red lines.
The remaining 13 AGR→CGU mutations were not observed, suggesting a codon substitution frequency of less than our detection limit of 1% of the bacterial population (Materials & Methods, Table S6). These "recalcitrant codons" were assumed to be deleterious or non-recombinogenic and were triaged into a troubleshooting pipeline for further analysis (Figure 1). Interestingly, all except for one of the thirteen recalcitrant codons were co-localized near the termini of their respective genes, suggesting the importance of codon choice at these positions — seven were at most 30 nt downstream of the start codon, while five were at most 30 nucleotides (nt) upstream of the stop codon (Figure 2A, lower panel, Table S8). Due to our unbiased design strategy, we anticipated that several AGR→CGU mutations would present obvious design flaws such as introducing non-synonymous mutations (two instances) or RBS disruptions (four instances) in overlapping genes. For example, ftsI_AGA1759 overlaps the second and third codons of murE, an essential gene, introducing a missense mutation (murE D3V) that may impair fitness. Replacing ftsI_AGA with CGA successfully replaced the forbidden AGA codon while conserving the primary amino acid sequence of MurE with a minimal impact on fitness (Figure 3A, Table S6). Similarly, holB_AGA4 overlaps the upstream essential gene tmk, and replacing AGA with CGU converts the tmk stop codon to Cys, adding 14 amino acids to the C-terminus of tmk. While some C-terminal extensions are well-tolerated in E. coli (37), extending tmk appears to be deleterious. We successfully replaced holB_AGA with CGC by inserting three nucleotides comprising a stop codon before the holB start codon. This reduced the tmk/holB overlap, and preserved the coding sequences of both genes (Figure S2A).
Figure 2. Analysis of attempted AGR→CGU replacements.
(A) AGR recombination frequency (MASC-PCR, n=96 clones per cell population) was plotted versus the normalized ORF position (residue number of the AGR codon divided by the total length of the ORF). Failed AGR→CGU conversions are indicated using vertical red lines below the x-axis. (B) Doubling time of strains in the C123 lineage in LBl media at 34 °C was determined in triplicate on a 96-well plate reader. Colored bars indicate which set of codons was under construction when a doubling time was determined (coloring based on Figure 1). Each data points represent different stages of strain construction. Alternative codons were identified for 13 recalcitrant AGR codons in our troubleshooting pipeline, and the optimized replacement sequences were incorporated into the final strain (gray section at right, labeled with a '*'), and the resulting doubling times were measured. Error bars represent standard error of the mean in doubling time from at least three replicates of each strain.
Figure 3. Examples of failure mechanisms for four recalcitrant AGR replacements.
Wild type AGR codons are indicated in bold black letters, design flaws are indicated in red letters, and optimized replacement genotypes are indicated in green letters. (A) Genes ftsI and murE overlap with each other. An AGA→CGU mutation in ftsI would introduce a non-conservative Asp3Val mutation in murE. The amino acid sequence of murE was preserved by using an AGA→CGA mutation. (B) Gene secE overlaps with the RBS for downstream essential gene nusG. An AGG→CGU mutation is predicted to diminish the RBS strength by 97% (47). RBS strength is preserved by using a non-synonymous AGG→GAG mutation. (C) Gene ssb has an internal RBS-like motif shortly after its start codon. An AGG→CGU mutation would diminish the RBS strength by 94%. RBS strength is preserved by using an AGA→CGA mutation combined with additional wobble mutations indicated in green letters. (D) Gene rnpA has a defined mRNA structure that would be changed by an AGG→CGU mutation. The original RNA structure is preserved by using an AGG→CGG mutation. The RBS (green), start codon (blue) and AGR codon (red) are annotated with like-colored boxes on the predicted RNA secondary structures.
Additionally, the four remaining C-terminal failures included AGR→CGU mutations that disrupt RBS motifs belonging to downstream genes (secE_AGG376 for nusG, dnaT_AGA532 for dnaC, and folC_AGAAGG1249,1252 for dedD, the latter constituting two codonsj. Both nusG and dnaC are essential, suggesting that replacing AGR with CGU in secE and dnaT lethally disrupts translation initiation and thus expression of the overlapping nusG and dnaC (Figure 3B, S2B). Although dedD is annotated as non-essential (31), we hypothesized that replacing the AGR with CGU in folC disrupted a portion of dedD that is essential to the survival of EcM2.1 (E. coli K-12). In support of this hypothesis, we were unable to delete the 29 nucleotides of dedD that were not deleted by Baba et al. (31) and did not overlap with folC, suggesting that this sequence is essential in our strains. The unexpected failure of this conversion highlights the challenge of predicting design flaws even in well-annotated organisms. Consistent with our observation that disrupting these RBS motifs underlies the failed AGR→CGU conversions, we overcame all four design flaws by selecting codons that conserved RBS strength, including a non-synonymous (Arg→Gly) conversion for secE.
These lessons, together with previous observations that ribosomes pause during translation when they encounter ribosome binding site motifs in coding DNA sequences (20), provided key insights into the N-terminal AGR→CGU failures. Three of the N-terminal failures (ssb_AGA10, dnaT_AGA10 and prfB_AGG64) had RBS-like motifs either disrupted or created by CGU replacement. While pf_AGG64 is part of the ribosomal binding site motif that triggers an essential frameshift mutation inprfB (21, 38, 39), pausing-motif-mediated regulation of ssb and dnaT expression has not been reported. Nevertheless, ribosomal pausing data (20) showed that ribosomal occupancy peaks are present directly downstream of the AGR codons for ssb and absent for dnaT (Figure S3); meanwhile, unsuccessful CGU mutations were predicted to weaken the RBS-like motif for prfB and ssb and strengthen the RBS-like motif for dnaT (Figure 3C, S2C), suggesting a functional relationship between RBS occupancy and cell fitness. Consistent with this hypothesis, successful codon replacements from the troubleshooting pipeline conserve predicted RBS strength compared to the large predicted deviation caused by unsuccessful AGR→CGU mutations (Figure 4, y axis and comparison between orange asterisks and green dots). Interestingly, attempts to replace dnaT_AGA10 with either CGN or NNN failed—only by manipulating the wobble position of surrounding codons and conserving the arginine amino acid could dnaT_AGA10 be replaced (Figure S2C). These wobble variants appear to compensate for the increased RBS strength caused by the AGA→CGU mutation—RBS motif strength with wobble variants deviated 8-fold from the unmodified sequence, whereas RBS motif strength for AGA→CGU alone deviated 27-fold.
Figure 4. RBS strength and mRNA structure predict synonymous mutation success.
Scatter plot showing predicted RBS strength (y-axis, calculated with the Salis ribosome binding site calculator (47)) versus deviations in mRNA folding (x-axis, calculated at 37°C by UNAFold Calculator (41)). Small gray dots represent non-essential genes in E. coli MG1655 that have an AGR codon within the first 10 or last 10 codons. Large gray dots represent successful AGR→CGU conversions in the first 10 or last 10 codons of essential genes. Orange asterisks represent unsuccessful AGR→CGU mutations (recalcitrant codons) in essential genes. Green dots represent optimized solutions for these recalcitrant codons. The "safe replacement zone" (blue shaded region) is an empirically defined range of mRNA folding and RBS strength deviations, based on the successful AGR→CGU replacement mutations observed in this study. Most unsuccessful AGR→CGU mutations (Orange asterisks) cause large deviations in RBS strength or mRNA structure that are outside the "safe replacement zone." Genes holB and ftsI are two notable exceptions because their initial CGU mutations caused amino acid changes in overlapping essential genes. Gene folC corresponds to 2 AGRs. Arrows for four examples of optimized replacement codons (ftsA, folC, rnpA, rpsJ) show that deviations in RBS strength and/or mRNA structure are reduced. Arrows are omitted for the remaining 8 optimized replacement codons so as to increase readability.
In order to better understand several remaining N-terminal failure cases that did not exhibit considerable RBS strength deviations (rnpA_AGG22, ftsA_AGA19, fTr_AGA16, and rpsJ_AGA298), we examined other potential nucleic acid determinants of protein expression. Based on the observation that mRNA secondary structure near the 5' end of Open Reading Frames (ORFs) strongly impacts protein expression (12), we found that these four remaining AGR→CGU mutations changed the predicted folding energy and structure of the mRNA near the start codon of target genes (Figure. 3D, S4). Successful codon replacements obtained from degenerate MAGE oligos reduced the disruption of mRNA secondary structure compared to CGU (Figure 4, green dots). For example, rnpA has a predicted mRNA loop near its RBS and start codon that relies on base pairing between both guanines of the AGG codon to nearby cytosines (Figure 3D, S5A). Importantly, only AGG22CGG was observed out of all attempted rnpA AGG22CGN mutations, and the fact that only CGG preserves this mRNA structure suggests that it is physiologically important (Figure 3D, S5B-C). In support of this, we successfully introduced a rnpA AGG22CUG mutation (Arg→Leu) only when we changed the complementary nucleotides in the stem from CC (base pairs with AGG) to CA (base pairs with CUG), thus preserving the natural RNA structure (Figure S5D) while changing both RBS motif strength and amino-acid identity. Our analysis of all four optimized gene sequences showed reduced deviation in computational mRNA folding energy (computed with UNAFold(40)) compared to the unsuccessful CGU mutations (Figure 4, x-axis orange asterisks and green dots). Similarly, predicted mRNA structure (computed with a different mRNA folding software: NUPACK(41)) for these genes was strongly changed by CGU mutations and corrected in our empirically optimized solutions (Figure S4).
Troubleshooting these 13 recalcitrant codons revealed that mutations causing large deviations from natural mRNA folding energy or RBS strength are associated with failed codon substitutions. By calculating these two metrics for all attempted AGR→CGU mutations, we empirically defined a safe replacement zone (SRZ) inside which most CGU mutations were tolerated (Figure 4, shaded area). The SRZ is defined as the largest multi-dimensional space that contains none of the mRNA folding energy or RBS strength associated AGR→CGU failures (Figure 4, red asterisks). It comprises deviations in mRNA folding energy of less than 10% with respect to the natural codon and deviations in RBS-like motif scores of less than a half log with respect to the natural codon, providing a quantitative guideline for codon substitution. Notably, the optimized solution used to replace the 13 recalcitrant codons always exhibited reduced deviation for at least one of these two parameters compared to the deviation seen with a CGU mutation. Furthermore, solutions to the 13 recalcitrant codons overlapped almost entirely with the empirically-defined SRZ. These results suggest that computational predictions of mRNA folding energy and RBS strength can be used as a first approximation to predict whether a designed mutation is likely to be viable. Developing in silico heuristics to predict problematic alleles streamlines the use of in vivo genome engineering methods such as MAGE to empirically identify viable replacement codons. Therefore, these heuristics reduce the search space required to redesign viable genomes, raising the prospect of creating radically altered genomes exhibiting expanded biological functions.
Once we had identified viable replacement sequences for all 13 recalcitrant codons, we combined the successful 110 CGU conversions with the 13 optimized codon substitutions to produce strain C123, which has all 123 AGR codons removed from all of its annotated essential genes. C123 was then sequenced to confirm AGR removal and analyzed using Millstone, a publicly available genome resequencing analysis pipeline (42). Two spontaneous AAG (Lys) to AGG (Arg) mutations were observed in the essential genes pssA and cca. While attempts to revert these mutations to AAG were unsuccessful—perhaps suggesting functional compensation—we were able to replace them with CCG (Pro) in pssA and CAG (Gln) in cca using degenerate MAGE oligos. The resulting strain, C123a, is the first strain completely devoid of AGR codons in its annotated essential genes (Sequences available online). Although some AGR codons in non-essential genes could unexpectedly prove to be difficult to change, our success at replacing all 123 instances of AGR codons in essential genes provides strong evidence that the remaining 4105 AGR codons can be completely removed from the E. coli genome, permitting the unambiguous reassignment of AGR translation function (23).
Kinetic growth analysis showed that the doubling time increased from 52.4 (+/− 2.6) minutes in EcM2.1 (0 AGR codons changed) to 67 (+/− 1.5) minutes in C123a (123 AGR codons changed in essential genes) in lysogeny broth (LB) at 34 °C in a 96-well plate reader (See Materials and Methods). Notably, fitness varied significantly during C123 strain construction (Figure 2B). This may be attributed to codon deoptimization (AGR→CGU) and compensatory spontaneous mutations to alleviate fitness defects in a mismatch repair deficient (mutS-) background. Overall the reduced fitness of C123a may be caused by on-target (AGR→CGU) or off-target (spontaneous mutations) that occurred during strain construction. In this way, mutS inactivation is simultaneously a useful evolutionary tool and a liability. Final genome sequence analysis revealed that along with the 123 desired AGR conversions, C123a had 419 spontaneous non-synonymous mutations not found in the EcM2.1 parental strain (Figure S10). Of particular interest was the mutation argU_G15A, located in the D arm of tRNAArg (argU), which arose during CoS-MAGE with AGR set 4. We hypothesized that argU_G15A compensates for increased CGU demand and decreased AGR demand, but we observed no direct fitness cost associated with reverting this mutation in C123, and argU_G15A does not impact aminoacylation efficiency in vitro or aminoacyl-tRNA pools in vivo (Figure S6). Consistent with Mukai et al. and Baba et al. (25, 31), argW (tRNAArg ccu; decodes AGG only) was dispensable in C123a because it can be complemented by argU (tRNAArgUCU; decodes both AGG and AGA). However, argU is the only E. coli tRNA that can decode AGA and remains essential in C123a probably because it is required to translate the AGR codons for the rest of the proteome (23).
To evaluate the genetic stability of C123a after removal of all AGR codons from all the known essential genes, we passaged C123a for 78 days (640 generations) to test whether AGR codons would recur and/or whether spontaneous mutations would improve fitness. After 78 days, no additional AGR codons were detected in a sequenced population (sequencing data available at https://github.com/churchlab/agr_recoding) and doubling time of isolated clones ranged from 22% faster to 22% slower than C123a (n=60).
To gain more insight into how local RBS strength and mRNA folding impact codon choice, we performed an evolution experiment to examine the competitive fitness of all 64 possible codon substitutions at each of AGR codons (Table S2). While MAGE is a powerful method to explore viable genomic modifications in vivo, we were interested in mapping the fitness cost associated with less-optimal codon choices, requiring codon randomization depleted of the parental genotype, which we hypothesized to be at or near the global fitness maximum. To do this, we developed a method called CRAM (Crispr-Assisted-MAGE). First, we designed oligos that changed not only the target AGR codon to NNN, but also made several synonymous changes at least 50 nt downstream that would disrupt a 20 bp CRISPR target locus. MAGE was used to replace each AGR with NNN in parallel, and CRISPR/cas9 was used to deplete the population of cells with the parental genotype. This approach allowed exhaustive exploration of the codon space, including the original codon, but without the preponderance of the parental genotype. Following CRAM, the population was passaged 1:100 every 24 hours for six days, and sampled prior to each passage using Illumina sequencing (see Table S2 & Figure 5).
Figure 5. Codon preference of 14 N-terminal AGR codons.
CRAM (Crispr-Assisted MAGE) was used to explore codon preference for several AGR codons located within the first 10 codons of their CDS. Briefly, MAGE was used to diversify a population by randomizing the AGR of interest, then CRISPR/Cas9 was used to deplete the parental (unmodified) population, allowing exhaustive exploration of all 64 codons at a position of interest. Thereafter codon abundance was monitored over time by serially passaging the population of cells and sequencing using an Illumina MiSeq. The left y-axis (Codon Frequency) indicates relative abundance of a particular codon (stacked area plot)). The right y-axis indicates the combined deviations in mRNA folding structure (red line) and internal RBS strength (blue line) in arbitrary units (AU) normalized to 0.5 at the initial timepoint. 0 means no deviation from wild type. The horizontal axis indicates the experimental time point in hours at which a particular reading of the population diversity was obtained. Genes bcsB and chpS are non-essential in our strains and thus serve as controls for AGR codons that are not under essential gene pressure.
Sequencing 24 hours after CRAM showed that all codons were present (including stop codons) (Figure S7), validating the method as a technique to generate massive diversity in a population. All sequences for further analysis were amplified by PCR with allele-specific primers containing the changed downstream sequence. Subsequent passaging of these populations revealed many gene-specific trends (Figure 5, S7, S8). Notably, all codons that required troubleshooting (dnaT_AGA10, ftsA_AGA19, frr_AGA16, rnpA_AGG22) converged to their wild-type AGR codon, suggesting that the original codon was globally optimized. For all cases in which an alternate codon replaced the original AGR, we computed the predicted deviation in mRNA folding energy and local RBS strength (as a proxy for ribosome pausing) for these alternative codons and compared these metrics to the evolution of codon distribution at this position over time. We also computed the fraction of sequences that fall within the SRZ inferred from Figure 4 (see Methods). CRAM initially introduced a large diversity of mRNA folding energies and RBS strengths, but these genotypes rapidly converged toward parameters that are similar to the parental AGR values in many cases (Figure 5, overlays). Codons that strongly disrupted predicted mRNA folding and internal RBS strength near the start of genes were disfavored after several days of growth, suggesting that these metrics can be used to predict optimal codon substitutions in silico. In contrast, non-essential control genes bcsB and chpS did not converge toward codons that conserved RNA structure or RBS strength, supporting the conclusion that the observed conservation in RNA secondary structure and RBS strength is biologically relevant for essential genes. Interestingly, tilS_AGA19 was less sensitive to this effect, suggesting that codon choice at that particular position is not under selection. Additionally, the average internal RBS strength for the ispG populations converged towards the parental AGR values whereas mRNA folding energy averages did not, suggesting that this position in the gene may be more sensitive to RBS disruption rather than mRNA folding. Gene lptF followed the opposite trend.
Interestingly, several genes (lptF, ispG, tilS, gyrA and rimN) preferred codons that changed the amino acid identity from Arg to Pro, Lys, or Glu, suggesting that non-coding functions trump amino acid identity at these positions. Importantly, all successful codon substitutions in essential genes fell within the SRZ (Figure 6), validating our heuristics based on an unbiased test of all 64 codons. Meanwhile non-essential control gene chpS exhibited less dependence on the SRZ. Based on these observations, while global codon bias may be affected by tRNA availability (6, 43-45), codon choice at a given position may be defined by at least 3 parameters: (1) amino acid sequence, (2) mRNA structure near the start codon and RBS (3) RBS-mediated pausing. In some cases, a subset of these parameters may not be under selection, resulting in an evolved sequence that only converges for a subset of the metrics. In other cases, all metrics may be important, but the primary nucleic acid sequence might not have the flexibility to accommodate all of them equally, resulting in codon substitutions that impair cellular fitness.
Figure 6. RBS strength and mRNA structure predict codon preference of 14 N-terminal codon substitutions.
Scatter plot showing the results of the CRAM experiment (Figure 5). Each panel represents a different gene. The Y-axis represents RBS strength deviation (calculated with the Salis ribosome binding site calculator (47)) while the X-axis shows deviations in mRNA folding energy (x-axis, calculated at 37°C by UNAFold Calculator (41)). Codon abundance at the intermediate time point (t=72hrs, chosen to show maximal diversity after selection) is represented by the dot size. Green dots represent the WT codon. Blue dots represent synonymous AGR codons. Orange dots represent the remaining 58 non-synonymous codons, which may introduce non-viable amino acid substitutions. Black squares represent unsuccessful AGR→CGU conversions observed in the genome-wide recoding effort (Table 1, Figure 1). The "safe replacement zone" (blue shaded region) is the empirically defined range of mRNA folding and RBS strength deviations, based on the successful AGR→CGU replacement mutations observed in this study (Figure 3). Genes bcsB and chpS are non-essential in our strains and thus serve as controls for AGR codons that are not under essential gene pressure.
These rules were used to generate a draft genome in silico with all AGR codons replaced genome-wide, reducing by almost fourfold the number of predicted design flaws (e.g., synonymous codons with metrics outside of the SRZ) compared to the naive replacement strategy (Figure 7, Figure S9, Table S7, see Methods). Furthermore, predicting recalcitrant codons provides hypotheses that can be rapidly tested in vivo using MAGE. Successful replacement sequences can then be implemented together in a redesigned genome. Encouragingly, since all newly predicted design flaws occur in non-essential genes, they would be less likely to impact fitness unless (1) despite the "non-essential" annotation, the gene is actually essential or quasi-essential (i.e., inactivation would impair growth), or (2) the codon in a non-essential gene impacts the expression of a neighboring essential gene (e.g., impacts an RBS motif or RNA structure). While incorrect genome annotations can only be addressed empirically (as demonstrated with gene dedD), further analysis reveals that AGR codons in non-essential genes should rarely impact annotated essential genes. In E. coli MG1655, only three AGR codons in non-essential genes overlap with the initial mRNA and RBS motifs of essential genes, and at least one synonymous CGN codon is predicted to obey the SRZ for all three cases. Furthermore, even if all synonymous mutations were to disobey the SRZ, since disruption of non-essential gene function should not compromise viability, it is expected that non-synonymous mutations in non-essential genes would be viable as long as they conserve crucial motifs impacting expression of the essential gene. Importantly, we confirmed by MAGE that AGR→CGU codon replacement was possible for 2 of these 3 cases and that an alternative synonymous solution could be found in the remaining case (see Methods).
Figure 7. Predicting optimal replacements for AGR codons reduces the number of codons that are predicted to require troubleshooting.
(A) Empirical data from the construction of C123. 110 AGR codons were successfully recoded to CGU (green), and 13 recalcitrant AGR codons required troubleshooting (red, striped). (B) Predicted recalcitrant codons (codons for which no CGN alternatives fall within the SRZ in Figure 4) for replacing all instances of the AGR codons genome-wide. The reference genome used for this analysis had insertion elements and prophages removed (48) to reduce total nucleotides synthesized and to increase genome stability, leaving 3222 AGR codons to be replaced (see Methods). Our analysis predicts that replacing all instances of AGR with CGU would have resulted in 229 failed conversions ('Naive Replacement', red striped). However, implementing the rules from this work ('Informed Replacement') to identify the best CGN alternative reduces the predicted failure rate from 7.1% (229/3222), to 2.0% (64/3222AGR) of which only a small subset will have a direct impact on fitness due to their location in non-essential genes. In such cases, MAGE with degenerate oligos could be used to empirically identify replacement codons as we have demonstrated herein. Each specific synonymous CGN is identified with a unique shade of green and is labeled inside of its respective section.
To conclude, comprehensively removing all instances of AGR codons from all E. coli essential genes revealed 13 design flaws which could be explained by a disruption in coding DNA Sequence, RBS-mediated translation initiation, RBS-mediated translation pausing, or mRNA structure. While the importance of each factor has been reported, our work systematically explores to what extent and at what frequency they impact genome function. Furthermore, our work establishes quantitative guidelines to reduce the chance of designing non-viable genomes. Although additional factors undoubtedly impact genome function, the fact that these guidelines captured all instances of failed synonymous codon replacements (Figure 4) suggests that our genome design guidelines provide a strong first approximation of acceptable modifications to the primary sequence of viable genomes. These design rules coupled with inexpensive DNA synthesis will facilitate the construction of radically redesigned genomes exhibiting useful properties such as biocontainment, virus resistance, and expanded amino acid repertoires (46).
This work was supported by the US Department of Energy [DE-FG02-02ER63445]; by the US Defense Advanced Research Projects Agency [N66001-12-C-4211 to F.J.I. and D.S.]; by the National Institute of General Medical Sciences [GM22854-42 to D.S.]; by a US Department of Defense NDSEG Fellowship (to M.J.L. and G.K.); by a US National Science Foundation Graduate Research Fellowship (to D.B.G.); by the Lynch Foundation (to M.M.L.); by an Amazon AWS in Education Grant Award (to G.K.); and by the Arnold & Mabel Beckman Foundation and DuPont, Inc. (to F.J.I.). Funding for open access charge: US Department of Energy [DE-FG02-02ER63445]. Stephanie Yaung generously provided CRISPR plasmids and technical support. A patent application has been filed by Harvard University relating to aspects of the work described in this manuscript. In the interests of transparency, we wish to mention that G.M.C. is a founder of Enevolv Inc. and Gen9bio (neither of which was involved in this study). Other potentially relevant financial interests are listed at http://arep.med.harvard.edu/gmc/tech.html.
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In 2003 the historic Kitty Hawk Pier was smashed by Hurricane Isabel after it had just undergone $300,000 in renovations. Purchased in 2006 by Kitty Hawk's new Hilton Garden Inn, the pier was remodeled and then reopened in 2008. The newly renovated Pier House now serves as a unique setting for wedding receptions and other social events, enhanced by the environment and tradition of the Outer Banks. But the pier itself still remains open to the public for fishing -with a purchased pass.
Sitting on high pilings over the Atlantic Ocean, the Pier House is 2200 square feet and features a beautiful natural wood interior with high ceilings and rafters and seating for roughly 125 guests. The bride and groom can have their wedding ceremony right on the beach, their first dance on the pier deck, a reception inside the Pier House and their honeymoon at the Inn, without having to travel anywhere.
The Inn, which sits just behind the pier is staffed with wedding coordinators who will make all the arrangements.
Weekly and yearly passes for fishing can be purchased at the front desk of the Inn for $30 and $125.00. Though the pier was shortened by a more recent hurricane, it's said to still be a good site for catching spot and drum. Kitty Hawk Pier and Pier House are open year round for fishing and special events.
Call 252-261-1290 for current hours.


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@implementation GitHubModels

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redpajama_set_name: RedPajamaGithub


SAN FRANCISCO – January 8, 2017 – EyeQue™, developers of award-winning, affordable vision technologies today announced that the EyeQue Insight™ is now available for purchase at eyeque.com. Having successfully exited a Kickstarter campaign designed to elicit consumer feedback on the new at-home vision platform, the Insight has now been added to the EyeQue online store and will begin shipping to backers and customers in March 2018.
EyeQue intelligent vision solutions empower anyone to take an active role in their vision care.
The EyeQue Insight is the only binocular smartphone-based vision test to accurately assess distance vision, creating a cloud-based digital vision history.
Combined with the award-winning EyeQue Personal Vision Tracker™, the two form an at-home vision screening power pack. Insight tests for 20/20 vision in under 3 minutes – with or without wearing corrective lenses. The Personal Vision Tracker can be used when results of the Insight show less than 20/20 results to determine refractive error, the numbers used to order corrective glasses online. Both products can be shared with anyone with an active annual EyeQue subscription ($4.99 per year) making it a great value for families, organizations, and charities.
The EyeQue Insight is the world's first gamified 20/20 screener, combining a binocular viewer, a mobile application, and cloud-based service. Low-cost, rugged, and convenient, the Insight is ideal for families, school administrators, group organizations, and charities. Insight is suited for international use with support for 20/20, 6/6, and other standardized units.
One EyeQue Insight can be shared with many people, making it a value for families and suitable for testing large groups.
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Pakistan v England: Women's side to tour Pakistan for first time in October
The two sides last met at the 2020 T20 World Cup in February, with England winning by 42 runs in Australia
England women will visit Pakistan for the first time when they play a Twenty20 and one-day series in October.
The two T20s will be played on 14 and 15 October in Karachi, as part of a double header alongside the men's team.
Heather Knight's side will then stay in the city for one-day international matches on 18, 20 and 22 October.
"This is another important step in our history and journey," said England and Wales Cricket Board managing director of women's cricket Clare Connor.
"Not only will this be a cricket tour that provides valuable competitive opportunity, it should also serve as another powerful message as we drive forward our equality ambitions for more women and girls to derive empowerment through the sport."
The two sides last met at the 2020 T20 World Cup in February, with England winning by 42 runs in Australia.
In November it was announced the men's side will be touring Pakistan for the first time in 16 years.
"Women's T20 internationals taking place before the men's matches at the iconic National Stadium will mean more eyeballs will follow these matches than ever before," said Pakistan Cricket Board chief executive Wasim Khan.
"I am confident the women's tour will open up opportunities for their future tours that will further contribute in promoting women's cricket in Pakistan."
England women v Pakistan women fixtures
14: 1st IT20, National Stadium, Karachi
15: 2nd IT20, National Stadium, Karachi
18: 1st ODI, National Stadium, Karachi
20: 2nd ODI, National Stadium, Karachi
22: 3rd ODI, National Stadium, Karachi – BBC
Morocco approves AstraZeneca/Oxford COVID-19 vaccine – Minister
JUST IN: Work on 3000 ha Masvingo greenbelt starts

redpajama_set_name: RedPajamaCommonCrawl


text:
New Noise: Thandii
The beach based beauty talks us through her debut EP.
Moving to the beaches of Margate after losing a bet with a mate, rising star Thandii reflects that effortlessness of seaside life in her debut EP, "Forgetful". Full of soaring romantic synths and intricate drum beats, the four-track record is a dream-pop sensation, showing off Thandii's seductively smoky vocals and flawless songwriting technique.
Marking the beginning of what is bound to be the start of an exciting journey, we had a chat with Thandii to find out about the girl behind the gorgeous songs.
Hey Thandii, how's it going?
Pretty good thanks.
Do you remember the moment when you fell in love with music?
Yeah, I grew up with a lot of music in the house because my whole family are musicians and I've always loved it. It was hearing Joni Mitchell's Blue that was probably my first love. Although the style may be a few worlds away from where my sound is now, Joni was an artist I felt akin to and I listened to that album over and over.
You released your debut single, "Cameo", earlier this year. Can you tell us about the inspiration behind the track?
It's about someone who's struggling to take the lead in their life and what inspired it was recognising that you're not going to do anything if you don't be bold and brave and just get on with it.
You wrote it in Margate after moving there because you lost a bet. We've got to hear the story behind this…
Haha yeah, I got dared to move somewhere I'd never been before and I just took the dare very seriously. It was a stage where I felt like I was going round in circles in London. So I spent a couple of months staying up all night going out, and going over everything and one night got a little crazy and ended with the dare. And a few weeks later I went to Margate for the first time ever… the weather was shit, I got stuck in the rain and the sea was a murky brown …but somehow the British seaside charm won me over. I don't know how! So I looked at a flat.. and and started renting it straight away.
You've described your sound as "an attempt to put a female stamp on an otherwise male-dominated sound". Can you tell us a bit more about this?
There was a time where all the music I was getting excited about had a male vocal on it, and the stuff I was hearing that I liked with a female vocal always tended to be softer and dreamier. I naturally have a fairly soft voice and I didn't want this to dictate the music direction completely. I like bold drums, weird guitar sounds, interesting lyric placement and sweeping synths. So there was one day where I suddenly thought, why can't this be the sound?
Your debut EP, "Forgetful", came out on 26th May. What can we expect from it?
There are a lot of old synths and up front drums on the tracks. I'd probably say none of the tracks are shy. It's also really a snap shot of the last year where I feel like I've been sticking my neck out (musically) and not worrying about how that's going to be received.
What are you wanting people to take away from the EP?
I hope people find the sound exciting. I guess that as my first EP I want it to give an introduction to what's gonna come in the future from me.
What else do you have planned for this year?
There's a couple of live shows coming up, LeeFest is one of them. Apart from that I'm really excited to see how the year's going to pan out.
Places + Faces: London Pop-Up
7 Wonders: LGBT Cinema
We chat to Swedish-born, London-based singer-songwriter KLARA about her hometown's natural beauty and her upcoming debut album.
Singer-songwriter The Beach talks heartbreak and vulnerability upon the release of his new pop tune "Still In Love".
New Noise: Nick Hakim
Translating the most difficult time of her life into poignant pop bops, Ella Vos talks us through how music kept her fighting.
Producer Ukiyo talks his self-titled debut album and creating a whole project in lockdown.
Leah Kate's debut EP "Impulse" champions living your best life this summer.
Rising singer and producer Very Very talks her new single "Do Right", creativity and why she took a stand on human rights.
Meet R&B's acoustic guitar-playing newcomer Lotus Ma'at, as she talks inspirations, TikTok and her debut EP.
Multi-instrumentalist Karl Zine talks debut solo single "Greenscreen" and branching out from alt-pop duo RINNGS.
Argentinian-Australian newcomer Marcelo De La Vega talks debut single "Told You" and his upcoming EP "Conversation".
Y2K favourite and rising newcomer FAANGS talks creating empowering music and upcoming debut project TEETH OUT.
Australian pop star Tulliah drops captivating new single "Distant Dreams" and talks upcoming debut EP "Fre$h Hugs".
Urban folk pioneer Wassailer talks his debut album i, the bastard, stepping out on his own and working solo.
Model-turned-singer Lorenzo Doryon talks new single "Straight Into The Fire" and his upcoming debut EP "Paint The Night".
Montreal-hailed newcomer Zach Zoya talks his debut EP "Spectrum", working with UK R&B star Angel and that tellytubby-inspired video.
Dropping her soul-stirring debut single "Here Again" today, Sofia Lafuente talks her alt-pop sound and finding solace in music.
Rising newcomer Rosie Darling talks new single "Water Me Down", her ever-growing sound and what we can expect from her debut EP "Coping".
Rising Norwegian pop sensation SKAAR talks creating her stellar debut EP "The Other Side Of Waiting" and how being a health care worker changed her perspective.
Indie-rock group Two Weeks In Nashville talk their new single "Homeward Bound" and their upcoming debut album PAPER PLANES AND RAZOR BLADES.
Rising neo-soul artist Langa Mavuso talks the release of his heartwarming debut album LANGA and growing up in South Africa.


text:
Don't Be Fooled: "Medicare for All" Is Still a Threat
COMMENTARY Health Care Reform
Aug 28th, 2020 2 min read
COMMENTARY BY
Nina Owcharenko Schaefer
Senior Research Fellow, Health Policy
Nina Owcharenko Schaefer is well known as a champion of patient choice and robust competition in America's health insurance markets.
Both solutions double down on a government-run model that fails to recognize that more government is the problem, not the answer. BRENDAN SMIALOWSKI / Contributor / Getty Images
Noticeably absent from the Democrats' latest policy platform: any mention of Medicare for All. But don't be fooled.
The left has acknowledged that Obamacare has fallen short of cost and coverage expectations. Yet their solution is more of the same.
Public option proponents argue that the new government plan would increase choice, but in practice, the public option will have the opposite effect.
Noticeably absent from the Democrats' latest policy platform: any mention of Medicare for All. But don't be fooled. Efforts to push government-run health care are still a real threat.
Rather than fully and openly embracing a single-payer model, this new approach hides itself behind expanding Obamacare and adding to it a new government plan—the so-called public option. However, more government involvement in health care is still the problem.
Take Obamacare. It has led to higher cost and less choice. Premiums more than doubled—with a 125% increase in the cost of health insurance—from 2013 to 2018. In 2013, the year before Obamacare took effect, the average monthly premium for coverage in the individual market was $244 per month. By 2018, the cost was $550 per month.
As premiums increased, coverage options decreased. By 2019, there were nearly half as many insurers offering coverage through the government-run exchanges, and 77% of counties had two or fewer insurers offering plans on the government-run exchanges.
Higher costs and fewer options have driven many middle class families out of the insurance market altogether. Census data show that the number of uninsured increased from 2017 to 2018. Many individuals who qualify for assistance remain uninsured, and data from the Centers for Medicare and Medicaid Services show that from 2016 to 2018, enrollment in Obamacare among middle-class families who are not subsidized dropped by 40%.
As CMS Administrator Seema Verma notes, "As usual, critics of President Trump have been quick to blame the Administration's healthcare policies for this increase. But a review of key facts suggests the rising uninsured rate stems largely from Obamacare's failure to deliver affordable health insurance premiums and has created a new class of uninsured."
Reluctantly, the left has acknowledged that Obamacare has fallen short of cost and coverage expectations. Yet their solution is more of the same: Spend more taxpayer money on subsidies to insurance companies to cover up the cost increases and set up another government program—the public option—to cover up the lack of coverage options.
Both solutions double down on a government-run model that fails to recognize that more government is the problem, not the answer.
Increasing insurers' access to subsidies would mean big spending with little meaningful impact. As Doug Badger, a former presidential appointee on the National Economic Council, points out, "Although federal expenditures have increased and are projected to continue to increase in the future, the number of people benefiting from subsidies has been declining for three years.
The Congressional Budget Office (CBO) projects that both trends will persist over the next decade, with the number of subsidized beneficiaries dropping by roughly one-fourth, while annual spending on federal subsidies rises by more than one-third."
The introduction of yet another new government program—the so-called public option—also would do more harm than good. Public option proponents argue that the new government plan would increase choice, but in practice, the public option will have the opposite effect.
Under the public option scheme, the government plan—with its regulatory power—would set its prices below its private-market competitors.
Once the private competitors are squeezed out, the government plan would be the last and only coverage option available. Being the sole provider of health care, the government would assume full power and control over the health care dollars and decisions, leaving Americans at the mercy of the government for their health care.
The effect of both these policy prescriptions advances the Medicare for All agenda: spend more taxpayer money, eliminate all private coverage, and replace it with one government-run health plan.
The argument on the left is not a policy disagreement but a strategy disagreement. Medicare for All proponents may prefer an immediate and radical replacement of the health care system. But in the end, both approaches would lead to a troubling prescription: A government-run health care system for all.
This piece originally appeared in The Washington Times
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Tennessee Secures Authorization for Heritage-Backed Medicaid Reforms
No Free Care for All When It Comes to Government-Controlled Health Care
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← Greek debts cannot be repaid in euros!
Well worth following this blog! Good Luck Yanis Varoufakis!
As a fan of the BBC, I must say I was appalled by the depths of inaccuracy in the reporting underpinning this interview (not to mention the presenter's considerable rudeness). Still, and despite the cold wind on that balcony, it was fun!


You can also extrude any shape you want from the material design. It is complete offline installer standalone setup of SolidWorks 2013 32 Bit 64 Bit Setup For Windows. Equations Learn about linking part dimensions to equations and global variables. Only specific virtual environments are supported. You can remove these shortcuts using the Backspace key or reassign new shortcuts to these commands. Annotations The eDrawings Viewer does not support the option Always display text at the same size for SolidWorks documents in SolidWorks, select the Options tool, and then select Detailing for the Document Properties tab.
The model building actually starts with the 2Dimensional design and now it can be extended into the 3D model which is just beyond amazing. Anti-virus Supported by variety of anti-virus in the market. Sheet Metal Forming Tools Learn how to make sheet metal part design more efficient when you include forming tools from the Design Library. Only 64-bit operating systems and processors are supported. In addition, Autodesk recommends settings which allow Microsoft Windows to manage virtual memory, as needed. Autodesk makes no representations, warranties or other promises related to use of any product in any virtualization environment or with any virtualization technology.
A complete listing of certified graphics cards can be found on the. System Requirement Use this information to ensure you are always working with a SolidWorks supported and optimized system for hardware, operating system and Microsoft products. Note: Not all Autodesk product offerings may be virtualized. Microsoft's published shipment date for Windows 7 is Oct 22nd. Find and download graphics card drivers for your system to ensure system performance and stability. After you update, please come back and you will be able to view our site.
Help files excluding tutorials are available as Web help used by default or as local help files. Insert Part In the Insert Part PropertyManager, the Break link to original part option is not supported when inserting sheet metal parts created prior to SolidWorks 2013 into sheet metal parts created in SolidWorks 2013 or newer. SolidWorks 2013 Setup Free Download Latest Version For Windows. For the full list of qualified SolidWorks applications and to ensure successful installation and optimum performance, see. Propagate Gauge Table Edits to Dependent Sheet Metal Features When you create a parent sheet metal feature and select a gauge table in the PropertyManager for the first time, you can propagate the gauge table settings and corresponding bend allowance options to all dependent sheet metal features.
From the SolidWorks Tutorials Getting Started page, click Advanced Techniques and select the CircuitWorks Basics tutorial. Autodesk may make available information regarding use of products in virtualized environments. The Tab and Shift + Tab key shortcut combinations that hide and show the assembly component below the mouse pointer are now customizable. Where virtualization is authorized, all conditions and limitations specified in the applicable terms and conditions apply. Here is an overview of our current recommended hardware. If it is not already installed, you can.
To ensure up-to-date tutorials and local help, you must download the files. . Minimizing in-context references, utilizing large assembly modes and other performance tools will reduce the load on the processor. SolidWorks allows you to create 2D as well as 3D models effortlessly. The minimum requirements can be found on the.
Solution: Please make sure that you are installing the appropriate version of Autodesk® Inventor® software for your operating system. Here's where you can find out. Please upgrade to a newer browser to fully enjoy this site and the rest of the web. The archive server must have disk capacity to store these backup files. Only 1:1 text scaling is supported for SolidWorks part and assembly files opened in eDrawings. This is one of the key benefits of data management systems.
If you do not want to use the gauge table and bend allowance options for dependent sheet metal features, you can override these options individually by editing each of the child sheet metal features. SolidWorks 2011 products will run on Windows Vista and Windows 7 32 and 64 bit versions only. Some functionality can leverage and share the workload over. These can be in numerical it contains the area, care of circle, height, width etc. Features from the inserted part are not imported. The result is that some objects may appear slightly darker in SolidWorks 2013 compared to previous releases. These are physically connected to the motherboard, instead of using cables, but does come at a cost.
Note that a 64-bit version of Inventor cannot be installed on a 32-bit system and vice versa. Or log into the SolidWorks Customer Portal at and click 'Download Software and Updates' to access the update. From the SolidWorks Tutorials Getting Started page, click Advanced Techniques and select the Equations tutorial. For example, select 2013, and in a row for SolidWorks 2013 and Service Pack 2. To insert the part, clear Break link to original part. SolidWorks in another piece of software which let you create 3D model designs for your project.


The continued rebound in local and international share markets meant that my retirement savings balance increased $43,045 (4.47%) and my geared share portfolio increased $29,660. The continued deflation of the property bubble nationwide, and especially Sydney, meant that the gain in my share portfolio was somewhat offset by the valuation of our home decreasing by $4,198 (0.55%) during the month. The rate of decline in house prices appears to be moderating slightly, suggesting a normal house price cyclical decline rather than a 'crash' or deflation of a 'bubble'. Overall, my net worth recovered $68,732 (3.13%) during the month to $2,263,713. Still below my all time peak.
Funding my university studies and running costs of my start-up financial planning business continue to cost around $2,500 per month. I received my annual bonus last month (approx. $9K after tax), which will help with cash flow for a while. I setup a free business entry in the 'yellow pages' yesterday and was immediately called by a couple of charity fund-raisers this morning seeking sponsorship/advertising support this morning. I agreed to sponsor the local Neighbourhood Watch (via a $440 ad in the local youth support magazine they produce annually) but I will have to decline supporting any other local groups (at least until I have my first paying client!) I'm still working on the website for my business, but should get that up and running by the end of March.
A rebound in local and international share markets meant that my retirement savings balance increased $42,724 (4.64%) and my geared share portfolio increased $19,533. The continued deflation of the property bubble nationwide, and especially Sydney, meant that the gain in my share portfolio was more or less offset by the valuation of our home decreasing by $18,397 (2.37%) during the month. Overall, my net worth recovered $44,081 (2.05%) during the month to $2,194,981. Still well below my all time peak.
I've started using some of the credit available in my St George 'portfolio loan' facility to fund my financial planning expenses (the self-education costs of the Masters degree, and the monthly licencee fee and CRM/admin package fee for the basic module of 'Midwinter'). Until I start receiving some client fee income, this debt will accrue at a rate of about $2,430 per month. Using the one loan facility to fund the major business running costs and self-education costs will make it easy to keep track of the amount of (tax deductible) interest I pay.
My goal is to start generating business income during 2019, and to generate enough business income during 2020 to cover running costs ('break even'). Hopefully by 2021 the business will be profitable, or at least cover the running costs and related self-education costs (the Masters, then possible a MRes degree followed by a PhD in Financial Planning). We'll see how things pan out.
I received an unusual offer via email the other day - I could receive 20,000 Qantas Frequent Flyer points if I opened a superannuation account online with AustralianSuper using the provided link, and contributed $350 within six months.
Anyhow, I decided to take up the offer. It took about 5 minutes to complete the online application process, and the BPay details for making a contribution were available the following day when I logged in to my new account. I did a BPay of the $350 personal contribution the day after I opened the account, and the funds were reflected in my balance the next day (I invested 50% in Australian Shares and 50% in International Shares) - which had gone up to $351.25. I'll wait an see when the promised QFF points get credited, and then do a rollover application. All in all, a fairly quick and painless way to 'earn' an extra 20,000 QFF points.
As I'm trying to launch my business on a low-cost, DIY basis I registered my business domain name using Dotster but then want to host the website content using my existing personal hosting account with GoDaddy. So I had to set up a 'redirect' of the URL from the name servers at Dotster to the hosting at GoDaddy. Took a bit of fiddling around but it seems to be working OK.
The next step was to install Wordpress for that domain in GoDaddy, and install a suitable 'Theme' (free of course). I eventually picked 'Sydney' after dabbling with 'Tesseract' but not being able to get it to work properly. I had a bit of trouble getting the recommended plugins (Elementor and Sydney Tools) working until I eventually figured out that I needed to upgrade the version of PHP installed in my GoDaddy hosting from 5.2 to 5.4 (and eventually I went to 5.6 as it was needed by 'Appointment Booking Scheduler' by Weblizar that I'll use for my online client booking tool).
I now seem to have all the tools installed to create the website content I have (vaguely) in mind - so the next stage is to sketch out my sitemap and page layouts and to actually write my page content, pick some suitable (free) stock photos to use etc. I could waste tons of time fiddling around with this stuff (I already wasted time browsing through free stock photos to pick a suitable background image for my page 'header' background), so I'll have to be quite disciplined about working towards a website that is 'fit for purpose' rather than trying to make it an award-winning masterpiece.
I did a couple of days of 'induction training' in December (had to fly from Sydney to Perth for the training, as the AFSL company does their training there) and managed to get 'registered' with ASIC as a Financial Planner before 1 Jan 2019. Looking back over my records, I'd been enrolled in the DFP course on and off for about six years, so it was only the pending FASEA rule changes that prompted me to hurry up and finish off the DFP to I could get registered before the new work experience requirements came into effect.
Having completed the DFP I'm now enrolled in the ADFP ('Advanced' Diploma) with IIT, and I'm also doing a Master of Financial Planning (via distance education) through Western Sydney University. I aim to get the ADFP completed during 2019, while also setting up my business website, creating a marketing 'funnel' to prospect for potential clients, and (hopefully) starting to provide advice to some clients. I should be able to complete the Masters degree sometime in 2021. I might then do a MRes degree with the idea of eventually doing a PhD in financial planning at WSU (that should be a *lot* easier to complete part-time than the PhD I tried to do in astrophysics).
Fortunately the AFSL company I've signed on with provides considerable resources for their 'authorised representatives' in the form of documentation templates, feedback on strategies being considered for clients, and has a requirement for all SoA (Statements of Advice) to be prepared by a professional third party paraplanner service. They also 'vet' all SoAs produced by new planners, so there is considerable supervision and assistance provided. On the down side their monthly fee is over $1000 (plus they also retain 20% of all revenues until annual income reaches a certain threshold), and there is also a minimum monthly fee for the required CRM software. Then for each SoA there will be paraplanner and vetting fees of around $250. The fees charged by the AFSL company cover such things as PI (Professional Indemnity) insurance, annual compliance costs (monitoring CPD requirements are met and conducting an annual audit) and the processing client fees and commissions.
My initial plan is to slowly build up to servicing around one new client per month, by which point I should just about 'break even'. Fortunately I'll be running this business in the evenings and weekends, while still keeping my full-time 'day job', so there isn't any great need to become profitable in the short term. Initially my business losses will have to be 'carried forward' until eventually I'll be able to deduct them against my other (salary) income (once my assessable income as a sole trader reaches $20,000 pa).
Aside from all the usual 'business expenses', I will also need to keep track of how much time I spend working on my business in my 'home office'. I won't use the dedicated office area I've setup in my home for meeting with clients (I plan on meeting with clients in their homes), so I won't be able to claim a deduction for pro-rata 'occupancy expenses' such as rates, house insurance and so forth, but I will be able to claim a deduction (at the set rate of $0.45/hr) for 'running expenses'.
I also had to register for GST, so I've started using Reckon One (online accounting) to track things and help prepare my annual BAS statements. As I'm now registered for GST I'll also have to include any UberEats delivery income in my BAS returns.
Another negative month, with my geared share portfolio and retirement savings balances each dropping by more than $20K, while our house price estimate stabilized. Still waiting (and hoping) that the global stock market 'correction' is over, but it appears that there may be further weakness ahead. The Australian economy is still growing quite well, so there may soon be a buying opportunity. Our Superannuation is already fully invested in growth, but my gearing is currently very low, so I could use my available margin loan facilities to invest in the local stock market when it seems to have reached the 'bottom'. However, as I am currently funding around $1,200 per month for my Financial Planning registration fees (without any clients so far) from my wage cash flow, I don't really want to add margin loan interest payments at this stage.
- complete the four courses for the Master of Financial Planning degree I've enrolled in for 2019 (1 per quarter).


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iSoftBet goes live with Eyas Gaming
iSoftBet, part of IGT PlayDigital, has established a partnership with experienced operator Eyas, to provide the brand with its high-quality gaming services and products.
The agreement will see several of iSoftBet's high-quality titles and third-party gaming content go live across Eyas' network, allowing its customers to enjoy some of the most engaging gaming experiences on the market.
Founded in 2020 by a group of iGaming industry veterans, Eyas is a UK-facing operator with a well-rounded team comprising of IT development, marketing, and customer operations, backed by the Gauselmann Group – Germany's leading land-based slots manufacturer and arcade operator.
The deal further reinforces the impressive commercial success that iSoftBet has experienced in recent months, having established deals with experienced operators Lowen Play, Alphabet and Casino Interlaken.
Lars Kollind, Head of Business Development at iSoftBet, said: "Off the back of this comprehensive partnership, Eyas' customers are finally able enjoy our impressive proprietary games selection and third-party offering.
"Our pioneering games will now feature across Eyas' sites – including Merkur Slots and Merkur Casino in the UK – and reach new audiences, helping us on our mission to further awareness about our multifaceted portfolio."
Jack Anthony, Head of Product at Eyas, said: "We are thrilled with the deal that has been established, allowing us to offer iSoftBet's high-quality slot titles across our various sites.
"We expect our customers to take full advantage of the brand's efficient aggregation platform, which provides a vast selection of gaming products from a range of the industry's leading suppliers."
Related Topics:Head of Business Development at iSoftBetHead of Product at EyasIGT PlayDigitaliSoftBetJack AnthonyLars Kollind
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Here are this weeks latest slots releases compiled by European Gaming!
As they say, this phrase is meant to bring us extreme luck! So, let's hop into our most adorable slot game, put this superstitious tale to the test, and shout "Rabbits, Rabbits, Rabbits!" to help Mr. Rabbit find his darling Mrs. Rabbit. Will this phrase increase his charm and luck after all? This is a new 5-reel, 3-row game with 10 paylines. Among the symbols on the reels, you'll find rabbits, gem-stones, classy cocktails, an old car, and a bouquet of cabbage. The Wild acts as a dollar sign with bunny ears!
MGA Games presents Roma Plus, the premium version of Roma, one of the company's most emblematic products. The renewed version of the 3-reel classic – the second online slot game created by MGA Games – includes significant game changes and the latest technology making the game playable on mobile, the users preferred channel.
Evoplay has launched its latest release, Mega Greatest Catch Bonus Buy, a new twist on its hit slot, Mega Greatest Catch. Inspired by the original game, Mega Greatest Catch Bonus Buy is an upgrade on its predecessor, integrating all known Bonus Buy features within the game. The Bonus Buy allows players to purchase 10, 15 or 20 Free Spins, so the bigger wins are always within grasp.
Greentube, is kick-starting the year with its most magical release to-date, Manic Potions™. Players looking to unlock the mystery behind this 5-reel, 1024 ways to win slot, will discover a concoction of fantastic features including a respin bonus, Lock & Spin bonus with a Wincreasing™ mechanic, and a medley of mystic modifiers.
Relax Gaming, continues its 2023 content roll-out with its spinetingling new slot, Horror Hotel. Across a quiet graveyard, players are invited to check in to an old lodge where 6×6 high-volatility fun, an RTP of 96.18% and max win of 20,000x bet await. The game sees the return of the Mystery Symbol feature, introduced in Relax's Santa's Stack, which is activated on any win.
Lightning Box looks set to harvest another hit after returning to the farm for Stellar Cash Chicken Fox 5x Skillstar. This 3×5 reeler, which boasts 20-win lines, features the studio's thrilling Stellar Cash feature integrated within one of its most popular games. It will make its debut with Sky Betting and Gaming this week for a fortnight exclusive before a general release across the UK and Europe.
Instant wins are turned up to the max in Kalamba Games' fresh take on a classic slot with 9 Blazing Cashpots Megaways™. Fruits are synonymous in slots entertainment, being enjoyed by millions over the years, and Kalamba has strolled down memory lane to revisit a player favourite title, bringing with it some brand new flavour – and Megaways™ – in this ultimate homage to the traditional iconography.
Wizard Games has embarked on a journey across the desert in search of glorious rewards in latest release, Sultan's Palace Fortune. The three-reel slot is packed full of riches fit for a sultan, with glittering gems, wilds and scatters filling the reels. In addition, players can land cash prizes, launch the Sultan's Treasure feature, initiate Bonus Mini Games, and more for fortunes of fun and payouts.
Inspired Entertainment, Inc. is pleased to announce the launch of two vibrant new online and mobile games: Call of the Wild™ and Lightning Viking™.
Collect your sword and prepare for the pit in Play'n GO's latest action slot, Game of Gladiators: Uprising, the follow-up to their 2019 title, Game of Gladiators. Follow Spartacus as he embarks on a battle for freedom and fortune. Pitted against the fearsome Bellica by the evil Emperor Gaius only one gladiator will leave the ring victorious.
Spinomenal has returned to Ancient Egypt with its new game release, Lucky Jack – Book of Rebirth – Egyptian Darkness. In Lucky Jack's latest adventure, the story takes you back to a time when a heavy, all-consuming darkness fell upon Egypt, looming over the empire. Join Lucky Jack on his treacherous journey in an attempt to save the land and restore the sunlight through the mystical powers of the sacred Book of Rebirth.
Pragmatic Play, a leading content provider to the iGaming industry creates monstrous towers of symbols that award multipliers in latest release, Monster Superlanche™. Played across 6×5 reels, the title introduces a crew of colourful creatures that act as symbols within the slot, at least eight of these must be matching anywhere on the reels to award a win.
Yggdrasil and Reflex Gaming are looking to chase the pot of gold at the end of the rainbow in their latest collaboration, Double Lucky Mushrooms DoubleMax™. The 5×3, 20 payline slot is adapted from Reflex Gaming's original Lucky Mushrooms title. It has a distinctly Irish feel, with leprechaun hats, pots of gold, and colourful Gaelic runes acting as symbols, while the game's bonus round is enhanced by Yggdrasil's popular Game Engagement Mechanic (GEM) DoubleMax.
BF Games is serving up a treat in its latest mouth-watering release Sweet Reward. This satisfying 5×3 game is set in the colourful Candyland, packed with scrumptious features and sweet rewards. Landing three cream-filled scatter symbols triggers 12 free spins along with up to a x15 multiplier. The feature is served up on a different set of reels and can be retriggered, unlocking another 12 spins that are added to the current spin serving with the same multiplier.
Playson, the fast-growing digital entertainment supplier, has released Pirate Chest: Hold and Win – its first slot launch of 2023 which showcases a new Boost feature to award cash prizes instantly. This feature-rich game promises to be a huge hit for the supplier and comes on the back of a very successful 2022. In this newest title, Playson takes players on a journey to the seven seas with an opportunity to discover mega wins.
Wazdan, the innovative games provider, has released the fifth title in its top-performing and engaging Hot Slot™ series, Hot Slot™: 777 Stars. The straightforward and satisfying gameplay is brimming with classic traits and symbols that give off classic retro vibes. The inclusion of such a popular theme will see operators' metrics boosted significantly with the title set to follow its predecessors in becoming a player-favourite before long.
Stakelogic is launching players into the cosmos where out-of-this-world wins await in its latest video slot release, Star StaxxTM. This game is all about Multipliers and Stacked symbols with players being encouraged to strap themselves in for a wild, intergalactic big-win ride. Star Staxx takes the 5×3 reel format with 20 pay lines active. The game sits at the high end of the volatility scale with a max win Multiplier of 7,500x the player's bet.
Iron Dog Studio brings players a furiously fishy frenzy of a slot with Feeding Fury, a gorgeous addition to the 1X2 Network portfolio, with general release across the 1X2 Network on 26th January. And with the 'Feed or Die' bonus round offering a dynamic freespins experience with a powerful expanding collector feature, this game is really something to sink your teeth into.
Realistic Games has launched one of the fieriest releases of the year with Chilli Master. A highly volatile title that provides players with incrementally increasing multipliers and big win potential. Played across 5×3 reels this Latin American-inspired slot has symbols depicting tacos, burritos, avocados and more which must form a matching combination across the game's 20 paylines to award a win.
Red Rake Gaming has kicked off the new year by releasing a new 5×3 reel slot game, with a highly retro theme and two main characters – Captain Wild and the villain – who will battle it out to accumulate multipliers, increasing them on the "Intergalactic Wheel" and discovering "Mystery" symbols along the way.
Red Tiger has launched Blood Suckers™ Megaways™, a chilling revamp of the renowned NetEnt classic enhanced by the industry-changing MegawaysTM mechanics. This macabre game, where players are advised to hang tight onto their wooden stakes as they slay the vampires, comes in addition to great Red Tiger remakes of further successful heritage NetEnt titles, such as Gonzo's Quest™ Megaways™ and Piggy Riches™ Megaways™.
BGaming, a fast-growing iGaming provider, has announced the launch of its latest online slot Potion Spells with elements of Match-3 games. The magic-infused 7×7 slot offers players high volatility and 96.24% RTP, along with a variety of features including refilling reels, Spin and Match-3 elements.
World Pool, the globe's largest commingled horse racing pools created and powered by the Hong Kong Jockey Club (HKJC), will be extended further in 2023 with the addition of major fixtures, including Lightning Stakes Day in Australia, The Saudi Cup and the Irish Derby.
Following the successful introduction of World Pool Moment of the Year in 2022 in Great Britain and Ireland, this has now been extended to all global World Pool meetings and will reward stable staff with a total of over HK$1 million (approx. £103,000) in prizes, as well as a VIP trip to Hong Kong, thanks to additional funding from the HKJC, UK Tote Group, Racecourse Media Group (RMG) and Ascot Racecourse.
Starting on Saturday 28 January with Met Day at Kenilworth in South Africa, the first meeting where racing fans from over 20 countries will be able to bet into the World Pool, and ending on Melbourne Cup Day on 7 February in Australia, one World Pool Moment of the Day will be selected at each World Pool meeting to win a prize of £4,000 (HK$38,785).
Each winning groom from the season's 25 World Pool days will also enter the chance to be named World Pool Moment of the Year and win a VIP trip for four to Hong Kong for either the 2024 Hong Kong Derby or 2024 FWD Champions Day.
The 25 individual World Pool Moment of the Day winners will be narrowed down to a final four by a panel of global racing experts, before being voted on by the public for World Pool Moment of the Year, which will be revealed at the Hong Kong International Races at Sha Tin in December.
Last year's World Pool Moment of the Year winner was Alpinista and winning groom Annabel Willis, who lit up the Knavesmire at York Racecourse when landing the Group 1 Yorkshire Oaks before going on to win the Prix de l'Arc de Triomphe.
The Saudi Cup and Lightning Stakes to make World Pool debuts in Q1 2023
Lightning Stakes Day at Flemington on 18 February and Saudi Cup night in Riyadh on 25 February have been confirmed as the two new additions to the first quarter of World Pool races for 2023.
The opening quarter of the year now boasts five fixtures, with two visits to Meydan for Super Saturday (4 March) and Dubai World Cup Night (25 March), as well as the aforementioned Met Day (28 January) at Kenilworth.
Irish Derby and Lockinge Stakes among 18 UK and Irish World Pool events
For the first time, World Pool will be in operation at Newbury Racecourse in the UK with two races added to the World Pool season on Lockinge Stakes Day on 20 May, while Irish Derby Day is also set to feature amongst 18 UK and Irish events on 2 July. Coverage of Irish Derby Day will also include the Deutsches Derby at Hamburg-Horn, marking the first World Pool race from Germany.
In 2022, World Pool betting turnover hit a new high of HK$6 billion (approx. £622m), up from HK$4.8 billion (approx. £498m) in 2021, across the 21 racedays that were commingled, while Derby Day at Epsom recorded the single highest day of turnover with HK$66.1 million (approx. £6.8m) – *note: turnover is recorded in HK$, and approximate pound sterling figures are therefore based on current exchange rates.
Michael Fitzsimons, Executive Director Wagering Products of the Hong Kong Jockey Club, said: "We were absolutely thrilled with the results from our 21 World Pool meetings in 2022 and are excited to be extending that offering to 25 days this year.
"Having Australia and Saudi Arabia on the fixture list of 2023 World Pool meetings is another important step in the globalisation of World Pool, while adding some high-profile British and Irish meetings to the schedule is another exciting advancement.
"To complement our expanding list of World Pool meetings are the Moment of the Day and Moment of the Year initiatives, which for the first time will reward winning stable staff from all countries hosting World Pool races.
"Last year's launch of the competition in the UK and Ireland was met with great feedback from yards across the region and we look forward to opening that up to our World Pool days in South Africa, Australia, Saudi Arabia and Dubai."
Alex Frost, Chief Executive of the UK Tote Group, said: "We are looking forward to another World Pool season which now extends its reach across eight countries worldwide. For the UK and Ireland, it is welcome to see new additions to this year's schedule, with two races from Newbury's Lockinge Day added, along with Irish Derby Day, taking World Pool events hosted in the UK and Ireland to a total of 18.
"The ambitions for a global wagering platform are already being realised with over 20 pool betting operators now part of World Pool. Last year alone, the UK and Ireland saw record turnover of £521 million across 17 World Pool events, showcasing the best of our racing to a global audience, ensuring an improved value proposition for our own wagering customers and creating a more sustainable funding mechanism for horseracing in the future, with additional income for our racecourse partners.
"Our continued thanks go to our partners the Hong Kong Jockey Club, and in collaboration with RMG and Ascot, we are delighted to see Moment of the Day and Moment of the Year now extended to include all countries hosting World Pool races."
Martin Stevenson, Chief Executive Officer of Racecourse Media Group (RMG), which represents the media interests of The Jockey Club, Newbury, Goodwood, and York racecourses, said: "RMG is delighted to partner with Hong Kong Jockey Club, UK Tote and our racecourses to bring 11 high-quality British and Irish days to the innovative and important World Pool schedule.
"RMG looks forward to working with all the parties in growing both the sport's revenues and global appeal. We're also excited to see the World Pool Moment of the Day and Year awards increase in prize money and evolve into a global initiative for stable staff – the real unsung heroes of our sport."
Alastair Warwick, Acting Chief Executive Officer, Ascot Racecourse, said: "It's fantastic to see this truly global expansion of the meetings included in the World Pool for 2023. The continued growth of World Pool since launching at Royal Ascot in 2019 has been very impressive and hugely important for British racing. We look forward to being involved again across our major meetings this year."
Gregory Bortz, the Executive Chairman of Cape Racing, the operator of racing in the Western Cape of South Africa, said: "We are honoured to have WSB Met Day, one of the biggest and most prestigious days on the South African racing calendar, be included as a World Pool event. We believe the sight and sounds of nineteen of our finest equine athletes competing for this prestigious Grade 1 race, with the views and sounds of Cape Town playing out in the background, will make for an unforgettable experience for the countless racing enthusiasts around the globe betting into the World Pool.
"The Hong Kong Jockey Club represents the gold standard for racing operators around the world, and we at Cape Racing look forward to following their lead by ensuring our top-class racing provides the entertainment and turnover befitting of World Pool status."
Provisional World Pool events to be hosted on the following days in 2023:
Saturday 28: Met Day – Kenilworth Racecourse, South Africa
Saturday 18: Black Caviar Lightning Race Day – Flemington Racecourse, Australia
Saturday 25: Saudi Cup – King Abdulaziz Racecourse in Riyadh, Saudi Arabia
Saturday 4 March: Super Saturday – Meydan Racecourse, Dubai
Saturday 25 March: Dubai World Cup – Meydan Racecourse, Dubai
Other key fixtures will be finalised later in the year, but major meetings including Royal Ascot, King George Day, Goodwood Festival, York Ebor Meeting, British Champions Day and Melbourne Cup Day will also form part of World Pool 2023.
OnAir Entertainment™, the go-to provider of premium live casino content to operators, launched a new game, Blazing Dragon Tiger to their game portfolio on 26th January.
With its unique studio environment and table-felt animation, in Blazing Dragon Tiger players decide which spot to bet on and anticipate the winning side to be either Dragon, Tiger, or Tie, with the option of additional side bets.
Alongside the smooth gameplay and great UI visuals, the studio environment will bring greater value to customers by offering brandable studio elements. In addition, customisation by customers may also be in the configurable side bets and bet limits.
Real-time percentages will show how many users are betting on Dragon, Tiger, or Tie offering live predictions and statistics. Additionally, the option to zoom in and zoom out on specific roads will allow players to see better all-needed results on these roads.
Blazing Dragon Tiger will join the OnAir Entertainment's™ games portfolio with Standard Blackjack, Standard Roulette, Speed Roulette, Auto Roulette, 9 Pots of Gold™ Streamicon™, Eclipse Blackjack™, Card Matchup, and Lotus Speed Baccarat™.
OnAir Entertainment's™ games are designed to offer a real-life gaming experience thanks to world-class digital branding technology, professional game presenters, and sleek branded environments which offer an immersive experience for players.
Seval Kaya, Business Development Director at OnAir Entertainment™, added: "We are very happy to announce a new game in our portfolio with great animation features creating stands out in the industry. Furthermore, our customers can use the brand elements on this new gaming experience for even better customisation. We have achieved this wonderfully designed user interface thanks to everyone who was involved in bringing this game to life."


Located approximately 650 meters from Rockdale Train Station and walking distance to Rockdale Plaza, this top floor unit ticks all the boxes.
Situated in a fully renovated security building of 15 units this spacious unit features, two generous bedrooms both with built in wardrobes, a sun drenched combined living and dining area flowing out to a north east facing balcony, a modern kitchen with stone benchtops and quality appliances and a modern bathroom with a separate bath and shower. The property offers an internal laundry and a large single car lock up garage with ample storage and an automatic garage door.