Application of tandem expression module based on ribosome recovery factor antisense RNA

Abstract

The invention relates to the field of genetic engineering, in particular to application of a tandem expression module based on ribosome recovery factor antisense RNA, and the tandem expression module is used for biosynthesis of a regulatory element in cells and regulation of the upstream and downstream protein expression proportion of a tandem site under the condition of protein tandem expression. The combination of the antisense RNA sequence, the expression structure of the antisense RNA sequence and the linker sequence between the expression cassettes can effectively adjust the relative expression proportion of tandem expression upstream and downstream proteins, and is used for gene function circuits with related requirements and optimal expression proportion exploration for specific protein pairs.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology and is an application of a class of synthetic biology sequence element combinations, which have a regulatory function and can regulate the expression ratio of the downstream expression frame of the linker sequence relative to the upstream expression frame to meet the relative expression amount control requirements of different proteins under different background conditions and expression purposes. Background Art

[0002] Expression cassette tandem expression modules have been mentioned in many literatures, but no literature has systematically studied or summarized the regulation strategy of expression cassette tandem expression modules using connection sequences and their cell backgrounds.

[0003] Existing tandem expression modules based on expression frames basically do not involve the regulation of relative expression levels of tandemly expressed proteins. Most tandem expression technologies can only express different proteins in the tandem expression frame at specific relative expression levels and can only be used for specific experimental projects and specific experimental purposes. They lack adjustability and universality.

[0004] If a tandem expression module with adjustable relative expression can be found, the cost of searching for the optimal relative expression ratio can be significantly reduced. At the same time, the tandem expression module with adjustable relative expression can itself be used as a regulatory element in research and production related to synthetic biology. Summary of the invention

[0005] The present invention proposes a set of expression frame tandem expression modules including the application of the used connection sequence and the tandem expression protein relative expression regulation gene circuit. The combination of the connection sequence and the tandem expression protein relative expression regulation gene circuit can effectively regulate the relative protein expression amount of adjacent expression frames, thereby improving the universality of the expression frame tandem expression technology and laying a good foundation for further constructing a universal tandem expression technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] An application of a tandem expression module based on ribosome recycling factor antisense RNA can regulate the expression ratio of upstream and downstream proteins of the tandem site under tandem expression.

[0008] According to an embodiment of the present invention, the tandem expression module includes two independent expression regions, one of which is a transcription factor transcription region, including a constitutive promoter of the transcription factor fragment, which includes an RNA polymerase binding site and a transcription start site, a ribosome binding site, a transcription factor translation region and a terminator; the other region is a frr gene antisense RNA transcription region, including a transcription factor binding site, a frr antisense RNA transcription region and a terminator; and also includes a specific linker sequence for connecting different protein translation regions in tandem expression.

[0009] According to an embodiment of the present invention, the transcription factor is a protein that can induce transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce transcription of downstream genes before binding to a specific compound, but cannot induce transcription of genes downstream of the binding site after binding to the specific compound.

[0010] According to an embodiment of the present invention, the transcription factor is an arabinose transcription factor.

[0011] According to an embodiment of the present invention, the constitutive promoter of the transcription factor fragment is the constitutive promoter of the arabinose transcription factor.

[0012] According to an embodiment of the present invention, the tandem expression module is carried on a vector plasmid or a genome or on a free circular / linear mRNA.

[0013] According to an embodiment of the present invention, the vector plasmid is an Escherichia coli plasmid, including pet28a.

[0014] According to an embodiment of the present invention, the linker sequence is TGATG.

[0015] To facilitate those skilled in the art to understand the present invention, the main terms are explained as follows:

[0016] Gene circuit: A combination of DNA sequences that combines specific gene elements such as promoters, terminators, protein translation regions, and specific protein binding regions according to certain rules and arranges them at single sites or multiple sites to achieve specific functions.

[0017] Universality: The module can be used across species by means of codon optimization / replacement-related controlled elements.

[0018] Escherichia coli ribosome recycling factor frr expression cassette sequence:

[0019] GTGATTAGCGATATCAGAAAAGATGCTGAAGTACGCATGGACAAATGCGTAGAAGCGTT (omitted here, see the sequence list for details)

[0020] ACAAAGAAGCAGAACTGATGCAGTTCTGA (SEQ ID NO: 1)

[0021] pET28a plasmid full sequence:

[0022] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC (omitted here, see the sequence list for details)

[0023] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGG GTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 2)

[0024] J23119 Promoter:

[0025] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC→(SEQ ID NO: 3)

[0026] rrnB T1 terminator:

[0027] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC (SEQ ID NO: 4)

[0028] Arabinose promoter sequence (arrow indicates the direction of transcription initiation):

[0029] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT (omitted here, see the sequence list for details)

[0030] GATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ ID NO: 5) T7 terminator sequence (arrow indicates the direction of transcription termination):

[0031] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO: 6) Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0032] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACC

[0033] TGCATCGATTTATTATGACAACTTGACCGGCTACATCATTCACTTTTTCTTCACAACCGGCA

[0034] CGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTG (omitted here, see the sequence list for details)

[0035] CTTTTCATACTCCCGCCATTCAGAGAAGAAAACCAATTGTCCATATTGCATCAGACATTGC

[0036] CGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAA

[0037] AGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCT (SEQ ID NO: 7)

[0038] T7 terminator sequence (arrow indicates the direction of transcription termination):

[0039] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO: 8) HumanCALCA related gene sequence:

[0040] ATGGGCTTTCAGAAATTTAGCCCGTTTCTGGCGCTGAGCATTCTGGTGCTGCTGCAGGCG (omitted here, see the sequence list for details)

[0041] GCGTTTGGCCGCCGCCGCCGCGATCTGCAGGCGTGA (SEQ ID NO: 9)

[0042] Human ITGB1BP2 related gene sequences:

[0043] ATGGCGCTGGAACAGAAAGAACTGGATCAGGAACCGGGCGCGGGCCTGGATAGCCTGA (omitted here, see the sequence list for details)

[0044] TGGAAATGGATGAAGAAGAAAGCGATGATAGCGATGATGATCTGAGCTGGACCGAAGAAGAAGAAGAAGAAGAAGCGATGGGCGAATAG (SEQ ID NO: 10)

[0045] When a ribosome encounters a stop codon while transcribing an mRNA with a tandem expression frame, there may be two situations:

[0046] 1. The ribosome is dissociated into free ribosomal subunits by ribosome recycling factors, which bind to the RBS sequence on another mRNA to initiate a new round of translation.

[0047] 2. The ribosome continues to slide toward the 3' end of the mRNA and restarts translation after encountering the start codon of the new expression frame.

[0048] The beneficial effects of the present invention are as follows: the present invention proposes an interference sequence for the Escherichia coli ribosome recycling factor frr gene, and proposes a combined application method based on a linker sequence between frr and a specific expression frame.

[0049] The combination of the antisense RNA sequence and its expression structure designed by the present invention and the linker sequence between the expression frames can effectively regulate the relative expression ratio of the upstream and downstream proteins expressed in series, and can be used for gene function circuits with relevant needs and for exploring the optimal expression ratio for specific protein pairs; the design proposed by the present invention can be used as a regulatory element in research and production related to synthetic biology. It also provides the possibility for the subsequent construction of more complex gene function circuits. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] Instruments and equipment:

[0052] 1. PCR instrument

[0053] 2. Electrophoresis instrument

[0054] 3. Electrophoresis tank

[0055] 4. Ultra-low temperature refrigerator

[0056] 5. Biochemical incubator

[0057] 6. Autoclave

[0058] 7. Water bath

[0059] 8. Medical freezer

[0060] 9. Desktop centrifuge

[0061] 10. Pipette

[0062] 11. Oven

[0063] 12. Youmi ELISA reader

[0064] Reagents:

[0065]

[0066]

[0067] Biomaterials:

[0068] Related primers and synthetic sequences:

[0069] EP1: CATGTTGTTGCTCAGGTCGCAGACG (SEQ ID NO: 11)

[0070] EP2: GGAAGGGAAGAAAGCGAAAGGAGC (SEQ ID NO: 12)

[0071] EP3: CCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCG (SEQ ID NO: 13)

[0072] EP4: CGCTTTCTTCCCTTCCTTTCGCCCACGTTCGCCGGCTTTC (SEQ ID NO: 14)

[0073] EP5:GATCCGGAACATAATGGTGCAGG (SEQ ID NO: 15)

[0074] EP6: GCACTAAATCGGAACCCTAAAGG (SEQ ID NO: 16)

[0075] Commissioned synthesis sequence 1 (ITGB1BP2 first, then CALCA):

[0076] CATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT (omitted here, see the sequence list for details)

[0077] ATTATACCTAGGACTGAGCTAGCTGTCAAGCTCCTTTTCGCTTTCTTCCCTTC C (SEQ ID NO: 17)

[0078] Commissioned synthetic sequence 2 (first CALCA, then ITGB1BP2):

[0079] CATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT (omitted here, see the sequence list for details)

[0080] TGCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTCC (SEQ ID NO: 18)

[0081] Example 1 Construction of E. coli frr protein RNA interference tandem expression plasmid

[0082] (1) High-fidelity enzyme amplification and plasmid construction experiments:

[0083] 1. Primers EP001 and EP002 were designed to amplify two synthetic sequences (SEQ ID NO: 17 and SEQ ID NO: 18) according to the following system and PCR procedure. Primers EP003 and EP004 were designed to amplify the plasmid template pet28a plasmid according to the same system and PCR procedure.

[0084] system:

[0085]

[0086] PCR procedure:

[0087]

[0088] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products of the two synthetic sequences and the PCR product of the pet28a plasmid according to the instructions of the purification kit.

[0089] 3. According to the operation procedures in the instructions of the ready-to-use seamless cloning kit, the PCR purified products of the two synthetic sequences were connected with the PCR purified product of the pet28a plasmid.

[0090] 4. Follow the steps below to convert 5αChemically CompetentCell after overnight culture:

[0091] 1) Take 100 μL of competent cells melted on ice, add the two groups of ligation products in "3" respectively, mix gently (gently pipette or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0092] 2) Heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes.

[0093] 3) Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 40 min.

[0094] 4) Take an appropriate volume of the resuscitation solution and evenly spread it on LB solid culture medium containing 50ug / mL kanamycin, and culture it upside down in a 37°C incubator overnight.

[0095] (2) Construction result sequencing verification experiment:

[0096] The single colony obtained from the coated plate was sent for sequencing using primers EP005 and EP006 to verify the construction results.

[0097] Plasmid construction results:

[0098] Construction results of ITGB1BP2-CALCA expression plasmid:

[0099] TGGCGAATCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTCAT (omitted here, see the sequence list for details)

[0100] AGCTGACGCAGCGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 19)

[0101] CALCA-ITGB1BP2 expression plasmid construction results:

[0102] TGGCGAATCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTAT (omitted here, see the sequence list for details)

[0103] CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGG AT (SEQ IDNO: 20)

[0104] (3) Plasmid extraction experiment:

[0105] According to the sequencing results in (2), a single colony available on the transformation plate in (1) was selected and inoculated into 5 mL of LB liquid culture medium containing 50 ug / mL kanamycin for overnight activation, and plasmid extraction was completed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0106] Example 2 Effect of Frr protein RNA interference on protein expression of Escherichia coli MG1655 pet28a plasmid

[0107] (1) Plasmid transformation experiment

[0108] 1) The Escherichia coli MG1655 strain was made competent according to the procedure described in the super competent cell preparation kit.

[0109] 2) Take 100 μL of competent cells melted on ice, add the ITGB1BP2-CALCA expression plasmid and CALCA-ITGB1BP2 expression plasmid constructed in the technical solution, mix gently (gently pipette or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0110] 3) Heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes (do not shake the sample during standing on ice, otherwise it will reduce the transformation efficiency).

[0111] 4) Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics to the centrifuge tube and mix well.

[0112] 5) Take an appropriate volume of the resuscitation solution and evenly spread it on LB solid culture medium containing 50ug / mL kanamycin, and culture it upside down in a 37°C incubator overnight.

[0113] (2) Shake flask fermentation experiment

[0114] 1) Take the single colony obtained in (4) and activate it in 3 mL of LB solution containing 50 ug / mL kanamycin at 37°C overnight.

[0115] 2) 1% was inoculated into 6 250ml sterile conical flasks containing 30ml LB liquid medium containing 50ug / mL kanamycin. After culturing for 3h, sterilized arabinose was added at final concentrations of 0%, 1%, 2%, 3%, 4%, and 5% respectively to induce for 6h.

[0116] (3) Fermentation result processing

[0117] Take the fermentation results according to BeyoLytic TM The results were extracted using the bacterial active protein extraction reagent according to the instructions.

[0118] Dilute the extracted results according to *10, *100, *1000, *10000, *100000.

[0119] (4) Elisa result test

[0120] Take 1ul of the dilution result and follow the Human ITGB1BP2 (Integrinbeta-1-bindingprotein 2) ELISA Kit

[0121] Perform ELISA detection according to the Human CALCA (Calcitonin gene-related peptide 1) ELISA Kit instructions.

[0122] The results are as follows:

[0123]

[0124]

[0125]

[0126] Example 3

[0127]

[0128]

[0129]

[0130]

[0131] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An application of a tandem expression module based on ribosome recycling factor antisense RNA, characterized in that: The expression ratio of upstream and downstream proteins of the tandem site in the case of tandem expression can be regulated.

2. The use according to claim 1, characterized in that: The tandem expression module includes two independent expression regions, one of which is the transcription factor transcription region, including the constitutive promoter of the transcription factor fragment, which includes the RNA polymerase binding site and the transcription start site, the ribosome binding site, the transcription factor translation region and the terminator; the other region is the frr gene antisense RNA transcription region, including the transcription factor binding site, the frr antisense RNA transcription region and the terminator; and also includes a specific linker sequence that connects different protein translation regions in the tandem expression.

3. The use according to claim 2, characterized in that: The transcription factor is a protein that can induce transcription of downstream genes of the binding site after binding to a specific compound; or a protein that can induce transcription of downstream genes before binding to a specific compound but cannot induce transcription of downstream genes of the binding site after binding to the specific compound.

4. The use according to claim 3, characterized in that: The transcription factor is an arabinose transcription factor.

5. The use according to claim 2, characterized in that: The constitutive promoter of the transcription factor fragment is the constitutive promoter of the arabinose transcription factor.

6. The use according to claim 2, characterized in that: The tandem expression module is carried on a vector plasmid or a genome or on a free circular / linear mRNA.

7. The use according to claim 6, characterized in that: The vector plasmid is an Escherichia coli plasmid, including pet28a.

8. The use according to claim 2, characterized in that: The linker sequence is TGATG.

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