Method for synthesis, assembly and function test of artificial chloroplast genome of chlamydomonas reinhardtii

Patent metadata

Field	Value
Publication number	US-2025092407-A1
Application number	US-202218559847-A
Country	US
Kind code	A1
Filing date	Aug 12, 2022
Priority date	Aug 1, 2022
Publication date	Mar 20, 2025
Grant date	—

Abstract

A method for synthesis, assembly and function test of an artificial chloroplast genome of Chlamydomonas reinhardtii. Rational design has been carried out on the Chlamydomonas reinhardtii chloroplast genome for the first time, and total artificial synthesis of the Chlamydomonas reinhardtii chloroplast genome is proposed. By using totally chemically synthesized chloroplast genome segments, total chemical de novo synthesis and assembly of a chloroplast genome are achieved in a yeast-bacterium system. Then, a totally chemically synthesized chloroplast genome is transformed into Chlamydomonas cells to replace the original chloroplast genome, which works normally, and has been verified, fulfilling biological functions of the totally chemically synthesized chloroplast genome. According to the embodiments, the Chlamydomonas reinhardtii chloroplast genome is an efficient platform for carrying out synthetic biology operation.

First claim

1 - 10 . (canceled)

11 . A method for synthesis, assembly, and function test of an artificial chloroplast genome of Chlamydomonas reinhardtii , the method comprising de novo design, total chemical synthesis, assembly and function verification of an artificial chloroplast genome of Chlamydomonas reinhardtii , wherein a nucleotide sequence of the artificial chloroplast genome of Chlamydomonas reinhardtii is obtained by designing and modifying a nucleotide sequence of a wild-type Chlamydomonas reinhardtii chloroplast genome and adding a BAC vector backbone, a streptomycin resistance gene aadA, a paromomycin resistance gene aph VIII and an HA tag.

12 . The method of claim 11 , wherein the nucleotide sequence of the artificial chloroplast genome of Chlamydomonas reinhardtii has a full length of 221,372 bp.

13 . The method of claim 11 , wherein the artificial chloroplast genome of Chlamydomonas reinhardtii is divided into 44 primary segments when being de novo designed, each of the primary segments having 120 bp homologous recombination sequences at two ends.

14 . The method of claim 13 , wherein the primary segments are all synthesized through a chemical method.

15 . The method of claim 11 , wherein the BAC vector backbone has a length of 11,060 bp, and an insertion site is located at 205,535 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome; the streptomycin resistance gene aadA has a length of 1,630 bp, and an insertion site is located between 173,174 bp and 173,175 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome; the streptomycin resistance gene aadA has a length of 2,287 bp, and an insertion site is located between 71,064 bp and 71,065 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome; a 3×HA-1 tag is located behind atpI, and an insertion site is located between 170,786 bp and 170,787 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome; a 3×HA-2 tag is located behind rps4, and an insertion site is located between 33,292 bp and 33293 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome.

16 . The method of claim 11 , wherein the synthesis and assembly of the artificial chloroplast genome of Chlamydomonas reinhardtii comprises the following steps:
connecting the 44 primary segments of the de novo designed artificial chloroplast genome of Chlamydomonas reinhardtii to a pUC18 vector, respectively; co-transforming a BAC vector segment, a screening marker segment, a 7-35 bridging segment, a seg44 segment, segments seg35-seg43, a seg1 segment, and segments seg3-seg7 into a yeast BY4741, and screening by using a screening medium SC-URA to obtain a yeast strain-1 containing an intermediate plasmid 1; co-transforming a pRS415 vector segment and segments seg7-seg21 into a yeast BY4742, screening by using a screening medium SC-LEU, using a kanMX segment to replace a Met gene on a genome with a yeast strain obtained by screening for Met knockout, and using an SC-LEU+G418 medium for screening to obtain a yeast strain-2 containing an intermediate plasmid 2; co-transforming a pRS411 vector segment and segments seg22-seg35 into a yeast BY4741, and using a screening medium SC-MET for screening to obtain a yeast strain-3 containing an intermediate plasmid 3; hybridizing the yeast strain-2 and the yeast strain-3, and screening through an SC-LEU-MET plate; then carrying out spore production and spore division, and screening through an SC-LEU plate and an SC-MET plate; then hybridizing with a yeast SZU-JDY19 and a yeast SZU-JDY20; then hybridizing with the yeast strain-1 containing the intermediate plasmid 1, and screening through an SC-LEU-MET-URA plate; transforming an SZU-ZLP012 plasmid of a strain obtained by screening, and screening through an SC-URA-LEU-MET-HIS plate; and inducing I-SceI gene expression in the SZU-ZLP012 plasmid by using a galactose, cleaving an I-SceI site to linearize 3 intermediate plasmids, and then obtaining a yeast strain containing a complete artificial chloroplast genome of Chlamydomonas reinhardtii through homologous recombination in a yeast cell.

17 . The method of claim 16 , wherein a nucleotide sequence of the intermediate plasmid 1 has a full length of 92,477 bp and contains nucleotide sequences of 1-33,292 bp and 159,554 bp-205,535 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome, and a BAC backbone sequence is added at 205,535 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome; a nucleotide sequence of the intermediate plasmid 2 has a full length of 81,426 bp and contains nucleotide sequences of 28,513 bp-102,566 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome, and pRS406 vector sequences are connected at head and tail interfaces of genome nucleotide sequences; a nucleotide sequence of the intermediate plasmid 3 has a full length of 72,377 bp and contains nucleotide sequences of 97,668 bp-164,450 bp of the wild-type Chlamydomonas reinhardtii chloroplast genome, and pRS411 vector sequences are connected at head and tail interfaces of genome nucleotide sequences.

18 . The method of claim 11 , wherein the function test of the artificial chloroplast genome of Chlamydomonas reinhardtii comprises the following steps:
transferring the artificial chloroplast genome of Chlamydomonas reinhardtii into a chloroplast of Chlamydomonas reinhardtii CC 5168 by particle bombardment, and obtaining positive transformants by streptomycin screening, PCR and Southern Blot screening; carrying out homogenization screening on the positive transformants by using gradient streptomycin resistance concentration; and carrying out Western Blot experiment on the positive transformants for verification of protein expression; and detecting a growth curve of the positive transformants and photosynthesis of a repair mutant strain.

19 . The method of claim 18 , wherein detection segments for identification and positive screening of the positive transformants are aphVIII, BAC-seg44, and BAC-seg1, respectively.

20 . The method of claim 18 , wherein a probe used for Southern Blot verification of the positive transformants is obtained by amplification of aph VIII-F1/R1 and psaA-F/R primers, nucleotide sequences of the aph VIII-F1/R1 primer being shown in SEQ ID NO.1 and SEQ ID NO.2, and nucleotide sequences of the psaA-F/R primer being shown in SEQ ID NO.3 and SEQ ID NO.4.

Assignees

• Univ Shenzhen

Inventors

• Hu Zhangli
• Wang Chaogang
• Guo Chunli
• Zhang Guiying
• Jia Bin

Classifications

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N15/8214

  Plastid transformation · CPC title

• G01N2333/405

  from algae · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12N15/8207

  by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers · CPC title