CN108681658A

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

Info

Publication number: CN108681658A
Application number: CN201810493075.6A
Authority: CN
Inventors: 叶远浓; 张晓娅; 黄梦雅; 曾柱
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2018-10-19
Anticipated expiration: 2038-05-22
Also published as: CN108681658B

Abstract

The invention discloses a kind of algorithms of optimization foreign gene translation speed in Escherichia coli, include the following steps：All codon combinations situations of pairs of amino acid are all carried out sequence alignment, the preferred codons redefined using Escherichia coli SD similar structures effects by the effect for making gene translation minibreak firstly, for SD sequence similar structures with Escherichia coli SD sequences；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 the preferred codons after redefining.Present invention incorporates codon preference effect, the recycling effect of tRNA and SD sequence similar structures to make translation minibreak effect scheduling theory knowledge, designs comprehensive three kinds of effects, rational algorithms for Escherichia coli to be optimized to the codon service condition of foreign gene.

Description

A kind of algorithm of optimization foreign gene translation speed in Escherichia coli

Technical field

The present invention relates to gene fields, and in particular to a kind of calculation of optimization foreign gene translation speed in Escherichia coli Method.

Background technology

Currently, all being rested on single factor test mostly using the research for influencing gene translation about codon, mostly grind Study carefully mode be theoretical research in combination with corresponding experimental verification, single factor can be determined to gene by control variate method Translation efficiency is with the presence or absence of influence, and most of the work about gene translation speed is all the Preference about codon 's.To the combination effect of existing factor to probe into work then be fewer and fewer.

Invention content

To solve the above problems, the present invention provides a kind of calculations of optimization foreign gene translation speed in Escherichia coli Method.

To achieve the above object, the technical solution that the present invention takes is：

A kind of algorithm of optimization foreign gene translation speed in Escherichia coli, includes the following steps：

The effect for making gene translation minibreak firstly, for SD sequence similar structures, by all of pairs of amino acid Codon combinations situation all carries out sequence alignment with Escherichia coli SD sequences, wherein inevitable, there are one least similar password subgroups It closes, then thering are optimal codon combinations to correspond the combination of 400 kinds of pairs of amino acid；It is optimal by this 400 kinds Codon combinations, which sort 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, its is optimal for the most explanation of counts in this 400 kinds of optimal codons combinations Possibility is maximum, as the optimal codon of this kind of amino acid, that is, uses Escherichia coli SD similar structures effect again fixed The preferred codons of justice；

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 turned The preferred codons being changed to after redefining.

Present invention incorporates codon preference effect, the recycling effect of tRNA and SD sequence similar structures to make translation Minibreak effect scheduling theory knowledge designs comprehensive three kinds of effects, rational algorithms come to external source base for Escherichia coli The codon service condition of cause optimizes.

Description of the drawings

Fig. 1 is the algorithm pattern that effect is recycled in conjunction with preferred codons effect and tRNA.

Fig. 2 is the algorithm pattern that SD sequence similar structures make translation minibreak effect.

Fig. 3 is the innovatory algorithm figure that SD sequence similar structures make translation minibreak effect

Fig. 4 is a kind of flow chart of optimization foreign gene algorithm of translation speed in Escherichia coli of the embodiment of the present invention.

Fig. 5 is the protein expression spirogram of three kinds of GFT.

Specific implementation mode

In order to make objects and advantages of the present invention be more clearly understood, the present invention is carried out with reference to embodiments further It is described in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair It is bright.

Effect algorithm is recycled in conjunction with preferred codons effect and tRNA

(1) preferred codons are found according to the preferred codons effect of Escherichia coli first, specific method is according to big The tRNA of enterobacteria determines preferred codons using abundance, for same amino acid, corresponding to the higher tRNA of abundance Codon is the preferred codons of the amino acid；

(2) and then effect is recycled according to tRNA to optimize sequence using the preferred codons that previous step determines. Specific practice is that the codon that same amino acid can be expressed as in sequence is all converted to its corresponding preferred codons.Example As occurred this codon of ATC in sequence, so that it may to be turned this codon of ATC according to the codon preference in Escherichia coli The GTC codons that tRNA uses abundance relatively high are changed to,

(3) and so on whole section of sequence can be optimized.

Make the algorithm of gene translation minibreak effect based on SD sequence similar structures

By knowwhy it is known that ribosomal minibreak is can be with this during translation due to mRNA Caused by 3 ' the end hybridization of 16SrRNA on ribosomes.And the structure of the similar SD sequences in mRNA sequence is more, class Similitude like the structure and SD sequences of SD sequences is higher, then the number that ribosomes pauses is also more, the time of pause It is longer, cause the rate of gene translation also slower.So for the translation rate of optimization gene, we will be as far as possible Ground reduces the possibility that this phenomenon occurs.According to this conclusion, algorithm steps below are designed.

(1) first, two codons adjacent in sequence are taken out every time, is converted into corresponding two amino Acid；

(2) and then to all codons corresponding to both amino acid be combined, allow these codon combinations all Sequence alignment is carried out with the SD sequences of Escherichia coli, carries out calling ClustalW when sequence alignment, highest scoring person is most like , score the lowest is least similar.Because we it is desirable that with the SD sequences of Escherichia coli least similar sequence, institute Have using the minimum sequence of score, i.e., least similar codon combinations are as this optimization to adjacent codon

(3) and then again a pair adjacent codon adjacent to adjacent codon with this optimizes, in such processes successively Whole section of sequence is optimized, you can whole section of sequence of optimization.

(4) when comparing number one, No. second codon, at the same by number one, No. second codon exchange position again with The SD sequences of Escherichia coli are compared.Thus accomplish No. second codon after the SD sequence alignments with Escherichia coli Also the comparison of sequence has been carried out in the case of half part with first half, (1 length bonus point branch of branch finally is calculated to score 2 length, along with branch 2 adds the length of branch 3, it is all and divided by 2), so that it may to obtain combining number one, second Number codon and No. second, the similar effect result of third codon.

The present invention provides a kind of algorithms of optimization foreign gene translation speed in Escherichia coli, have combined above-mentioned calculation Method includes the following steps：

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 For the preferred codons after redefining.

Algorithm evaluation and verification：

We in genetic engineering it is most-often used to green fluorescence protein gene (GFP) carry out the proof of algorithm, pass through Genotype of the two sequences (translation speed is accelerated in an optimization, and an optimization slows down translation speed) with wild type after optimization It is compared and assesses.

Theoretical value is assessed：

What codon adaptation indexI (codon adaptation index, CAI) measured is some gene institute in organism It is an important indicator for reflecting codon preference with the degree that is consistent of codon and codon used in cance high-expression gene. And the important indicator in genetic engineering for reacting exogenous gene expression amount.We compare the CAI indexes of three kinds of codons,

Optimization improves the CAI of the GFP sequences of translation speed:0.877；

Wild type GFP CAI:0.611

Optimization reduces the CAI of the GFP sequences of translation speed:0.561；

As can be seen that the CAI after the algorithm optimization makes it

Experimental verification

We look for commercial company to synthesize corresponding sequence, are transferred in escherichia coli vector and observe according to the sequence of algorithm optimization It is transferred to rear expression quantity, the fast expression quantity within the unit interval of translation speed is bigger, and the results are shown in Figure 5：

The result shows that the algorithm can be applied to successfully in genetic engineering, foreign gene can be effectively improved/reduced big Translation speed in enterobacteria.

GFP-WT is the expressing quantity curve of wild type GFP；GFP-SLOW is the egg for the GFP that optimization reduces translation speed White expression quantity curve；GFP-FAST is the expressing quantity curve for the GFP that optimization improves translation speed；PBAD is blank control (no albumen).

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims

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.