CN108681658A - A kind of algorithm of optimization foreign gene translation speed in Escherichia coli - Google Patents

Abstract

The invention discloses an algorithm for optimizing the translation speed of exogenous genes in Escherichia coli, which includes the following steps: first, for the effect of short-term pause in gene translation due to the similar structure of the SD sequence, all codon combinations of paired amino acids are compared with Sequence comparison of E. coli SD sequences, using the preferred codons redefined by the similar structure effect of E. coli SD; then, combined with the tRNA recycling effect, all the codons that would be expressed as the same amino acid in the sequence were converted to the redefined preferred codons. The present invention combines theoretical knowledge such as the effect of codon preference, the recycling effect of tRNA, and the short pause effect of translation caused by the similar structure of the SD sequence, and designs a reasonable algorithm that integrates the three effects for Escherichia coli to extract the codons of foreign genes. Optimized for usage.

Description

An Algorithm for Optimizing the Translation Speed of Exogenous Genes in Escherichia coli

technical field

The invention relates to the field of genes, in particular to an algorithm for optimizing the translation speed of exogenous genes in Escherichia coli.

Background technique

At present, most of the studies on the influence of codon usage on gene translation remain on a single factor. Most of the research methods are theoretical research combined with corresponding experimental verification. The control variable method can determine whether a single factor has an impact on the translation efficiency of a gene. , and most of the work on gene translation rate is about codon bias. There is very little work exploring the combined effects of existing factors.

Contents of the invention

To solve the above problems, the present invention provides an algorithm for optimizing the translation speed of exogenous genes in Escherichia coli.

In order to achieve the above object, the technical scheme that the present invention takes is:

An algorithm for optimizing the translation speed of exogenous genes in Escherichia coli, comprising the following steps:

First of all, for the effect that the similar structure of the SD sequence makes gene translation pause temporarily, all the codon combinations of the paired amino acids are compared with the E. coli SD sequence, and there must be a most dissimilar codon combination, then for The 400 pairs of amino acid combinations have optimal codon combinations corresponding to them; these 400 optimal codon combinations are sorted out, and then the codons for each amino acid in the 400 optimal codon combinations Counting, for the codon of each amino acid, the one with the most number of counts among the 400 optimal codon combinations shows that it is most likely to be optimal, and it is used as the optimal codon for this amino acid, that is, using the large intestine Preference codons redefined by similar structure effects in Bacillus SD;

Then, combined with the tRNA recycling effect, all the codons that would be expressed as the same amino acid in the sequence were converted to the redefined preferred codons.

The present invention combines theoretical knowledge such as the codon bias effect, the recycling effect of tRNA, and the short translation pause effect caused by the similar structure of the SD sequence, and designs a reasonable algorithm that integrates the three effects for E. coli to extract the codons of foreign genes Optimized for usage.

Description of drawings

Figure 1 is a diagram of the algorithm combining the codon bias effect and the tRNA recycling effect.

Fig. 2 is an algorithm diagram of the short pause effect of translation caused by the similar structure of SD sequence.

Figure 3 is an improved algorithm diagram of the short pause effect in translation caused by the similar structure of the SD sequence

Fig. 4 is a flowchart of an algorithm for optimizing the translation speed of exogenous genes in Escherichia coli according to an embodiment of the present invention.

Fig. 5 is a graph showing the protein expression levels of three GFTs.

Detailed ways

In order to make the objects and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

An Algorithm Combining Preference Codon Effects and tRNA Cyclic Use Effects

(1) First find the preferred codon according to the preferred codon effect of E. coli. The specific method is to determine the preferred codon according to the abundance of tRNA used in E. coli. For the same amino acid, the codon corresponding to the tRNA with higher abundance The codon is the preferred codon of the amino acid;

(2) Then optimize the sequence using the preferred codons determined in the previous step according to the tRNA recycling effect. The specific method is to convert all the codons that will be expressed as the same amino acid in the sequence to their corresponding preferred codons. For example, if the codon of ATC appears in the sequence, the codon of ATC can be converted to the codon of GTC with relatively high abundance in tRNA according to the codon preference in E. coli.

(3) By analogy, the entire sequence can be optimized.

An Algorithm Based on the Similar Structure of SD Sequences to Make Gene Translation Pause Effect

From theoretical knowledge, we already know that the brief pause of the ribosome is caused by the hybridization of the mRNA with the 3' end of the 16S rRNA on the ribosome during translation. Moreover, the more SD sequence-like structures in the mRNA sequence, and the higher the similarity between the SD sequence-like structure and the SD sequence, the more times the ribosome pauses, and the longer the pause time, resulting in gene translation. The speed is also slower. Therefore, for optimizing the translation rate of genes, we should reduce the possibility of this phenomenon as much as possible. According to this conclusion, the following algorithm steps are designed.

(1) First, take out two adjacent codons in the sequence each time, and convert them into corresponding two amino acids;

(2) Then combine all the codons corresponding to these two amino acids, and make sequence comparisons between these codon combinations and the SD sequence of E. coli. When performing sequence comparisons, call ClustalW, and the one with the highest score is the most similar , the one with the lowest score is the least similar. Because what we need is the sequence least similar to the SD sequence of E. coli, the sequence with the lowest score, that is, the most dissimilar codon combination is used as the optimized sequence for the pair of adjacent codons

(3) Then optimize the adjacent codons adjacent to the pair of adjacent codons, and optimize the entire sequence sequentially in this way, so that the entire sequence can be optimized.

(4) When comparing the No. 1 and No. 2 codons, the exchange positions of the No. 1 and No. 2 codons were compared with the SD sequence of E. coli at the same time. In this way, the second codon is compared with the second half of the SD sequence of E. coli, and the sequence is also compared with the first half, and finally the score is calculated (branch 1 length plus branch 2 length, Add the lengths of branch 2 plus branch 3, and divide all the sums by 2), you can get the similar effect result combining the first and second codons and the second and third codons.

The present invention provides an algorithm for optimizing the translation speed of exogenous genes in Escherichia coli, which combines the above-mentioned algorithm and includes the following steps:

First of all, for the effect that the similar structure of the SD sequence makes gene translation pause temporarily, all the codon combinations of the paired amino acids are compared with the E. coli SD sequence, and there must be a most dissimilar codon combination, then for The 400 pairs of amino acid combinations have optimal codon combinations corresponding to them; these 400 optimal codon combinations are sorted out, and then the codons for each amino acid in the 400 optimal codon combinations Counting, for the codon of each amino acid, the one with the most number of counts among the 400 optimal codon combinations shows that it is most likely to be optimal, and it is used as the optimal codon for this amino acid, that is, using the large intestine Preference codons redefined by similar structure effects in Bacillus SD;

Then combined with the tRNA recycling effect, all the codons that would be expressed as the same amino acid in the sequence were converted into the redefined preferred codons.

Algorithm evaluation and verification:

We use the green fluorescent protein gene (GFP), which is most commonly used in genetic engineering, to verify the algorithm, and compare the two optimized sequences (one optimized to speed up translation, and one optimized to slow down translation) with the wild-type genotype. Compare and evaluate.

Evaluation of theoretical value:

Codon adaptation index (CAI) measures the degree of coincidence between the codons used by a certain gene in an organism and the codons used by highly expressed genes, and is an important indicator reflecting codon bias. It is also an important indicator used to reflect the expression level of exogenous genes in genetic engineering. We compared the CAI indices of the three codons,

Optimize the CAI of the GFP sequence that improves the translation speed: 0.877;

Wild-type GFP CAI: 0.611

Optimize the CAI of the GFP sequence that reduces the translation speed: 0.561;

It can be seen that the optimized CAI of the algorithm has reached the predetermined target.

Experimental verification

According to the sequence optimized by the algorithm, we found a commercial company to synthesize the corresponding sequence, and transferred it into the E. coli vector to observe the expression amount after transfer. The faster the translation speed, the greater the expression amount per unit time. The results are shown in Figure 5:

The results show that the algorithm can be successfully applied to genetic engineering, and can effectively increase/decrease the translation speed of exogenous genes in E. coli.

GFP-WT is the protein expression curve of wild-type GFP; GFP-SLOW is the protein expression curve of GFP that optimizes the translation speed; GFP-FAST is the protein expression curve of GFP that optimizes the translation speed; pBAD is the blank control ( protein-free).

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be It is regarded as the protection scope of the present invention.

Claims (1)

1. a kind of algorithm of optimization foreign gene translation speed in Escherichia coli, it is characterised in that：Include the following steps：

The effect for making gene translation minibreak firstly, for SD sequence similar structures, by all passwords of pairs of amino acid Sub-portfolio situation all carries out sequence alignment with Escherichia coli SD sequences, wherein least similar codon combinations there are one inevitable, So there are optimal codon combinations to correspond the combination of 400 kinds of pairs of amino acid；By this 400 kinds of optimal passwords Sub-portfolio, which sorts out, to be come, and is then counted to the codon of each amino acid in this 400 kinds of optimal codon combinations, for The codon of each amino acid, most explanation its optimal possibility of counts in this 400 kinds of optimal codons combination Property it is maximum, as the optimal codon of this kind of amino acid, i.e., redefined using Escherichia coli SD similar structures effects Preferred codons；

Then, effect is recycled in conjunction with tRNA, the codon that amino acid of the same race can be expressed as in sequence is all converted to Preferred codons after redefining.