Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120
蓝藻菌鱼腥藻属物种 PCC 7120中的靶标基因失活   

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Microbiology
Jun 2015

 

Abstract

Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N2-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an integrative vector to produce a cargo plasmid. Upon a single crossover (homologous recombination) between the cargo plasmid and the Anabaena chromosome, the endogenous target gene is disrupted by generating 3’- and 5’-deleted fragments. This gene inactivating protocol is based on an integrative vector pZR606 (Chen et al., 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Keywords: Genetic tools for bacteria (细菌的遗传工具), Genetic engineering (遗传工程), Integration vector method (整合载体的方法), Single cross-over method (单交叉法), Synthetic cyanobacteria (合成的蓝藻)

Materials and Reagents

  1. Petri dishes (Fisher Scientific, catalog number: FB0875713 )
  2. Glass flask (Fisher Scientific, catalog number: FB-500-50 )
  3. 1.5 ml centrifuge tubes (Fisher Scientific, catalog number: 02-682-550 )
  4. 15 ml conical centrifuge tubes (Fisher Scientific, catalog number: 05-527-90 )
  5. 50 ml conical centrifuge tubes (Fisher Scientific, catalog number: 12-565-270 )
  6. Anabaena sp. strain PCC 7120 (hereafter Anabaena 7120)
  7. Escherichia coli (E. coli) NEB10β [Δ(ara-leu) 7697 araD139 fhuAΔlacX74 galK16 galE15 e14-Φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL(StrR)rph spoT1Δ(mrr-hsdRMS-mcrBC); New England BioLabs, catalog number: C3019H ]
  8. E. coli HB101 [F-mcrB mrr hsdS20(rB-mB-) recA13, leuB6, ara-14, proA2, lacY1, galK2, xyl-5, mtl-1, rpsL20(SmR) glnV44 λ- ; Promega, catalog number: L2015 ]
  9. Conjugal plasmid pRL443 and helper plasmid pRL623 (Elhai et al., 1997)
  10. pZR606, an integrative vector for Anabaena 7120 (Chen et al., 2015). pZR606 is available upon request (GenBank, catalog number: KJ500179.1 )
  11. pZR670 (available upon request), a replicative vector in Anabaena 7120 (Xu et al., 205) [The map of pZR670 is provided (Figure S1).]
  12. LB broth (Sigma-Aldrich, catalog number: L3522-1KG )
  13. LB agar, used for growing E. coli (MP, catalog number: 100262 )
    Note: This brand of agar does not work for cyanobacteria.
  14. Agar, required for growing Anabaena 7120 and other cyanobacteria (Fisher Scientific, catalog number: A360-500 )
  15. Immobilon-NC transfer membrane (Millipore, catalog number: HATF08550 )
  16. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
  17. Chloramphenicol (Fisher Scientific, catalog number: BP904-100 )
  18. Erythromycin (Sigma-Aldrich, catalog number: E6376-25G )
  19. Kanamycin sulfate (Sigma-Aldrich, catalog number: K4000-25G )
  20. Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S9007-25G )
  21. MgSO4·7H2O (MP, catalog number: 194833 )
  22. CaCl2·2H2O (Fisher Scientific, catalog number: BP510-500 )
  23. NaCl (Fisher Scientific, catalog number: S271-1 )
  24. K2HPO4 (Fisher Scientific, catalog number: BP363-500 )
  25. KNO3 (Fisher Scientific, catalog number: BP368-500 )
  26. NaNO3 (Fisher Scientific, catalog number: BP360-500 )
  27. MnCl2·4H2O (Fisher Scientific, catalog number: M87-100 )
  28. Na2Mo4·2H2O (99% purity) (Acros Organics, catalog number: 206371000 )
  29. ZnSO4·7H2O (Fisher Scientific, catalog number: Z76-500 )
  30. CuSO4·5H2O (Fisher Scientific, catalog number: BP346-500 )
  31. H3BO3 (Fisher Scientific, catalog number: BP168-500 )
  32. NH4VO3 (Acros Organics, catalog number: 194910500 )
  33. CoCl2·6H2O (Fisher Scientific, catalog number: C371-100 )
  34. KOH (Fisher Scientific, catalog number: P250-500 )
  35. Na2EDTA·2H2O (Fisher Scientific, catalog number: BP120-500 )
  36. FeSO4·7H2O (Fisher Scientific, catalog number: I146-500 )
  37. Allen and Arnon medium plus nitrate: AA/8(N) (see Recipes)

Equipment

  1. Centrifuges (Beckman, model: Allegra X-15R ; Thermo Fisher Scientific, SorvallTM LegendTM, model: Micro17 )
  2. Innova-44R shaker equipped with continuous fluorescent light illumination (ca. 50-100 μE/m2 s) (Eppendorf, New BrunswickTM Innova®, model: 44R )
  3. Cyanobacteria Culture Room (constant at 30 °C) equipped with a digital temperature controlling system and Sylvania fluorescent light bulbs (F40CWX, 40W/4100K, T12)
    Note: Light shelves in cyanobacteria culture room are able to provide continuous light illumination (ca. 50-150 μE/m2 s).
  4. ESCO laminar flow cabinet (ESCO, Clean BenchAirstream® model: AHC-4B2 )
  5. Water bath sonicator (Bransonic® Ultrasonic Cleaner, model: 1510R-MT )
  6. Microscope, Olympus upright compound microscope (Olympus, model: AX70; BX53 )
  7. PCR thermal cycler (Bio-Rad Laboratories, TouchTM, model: C1000 )
  8. UV-Vis spectrophotometer (Thermo Fisher Scientific, model: GeneSyS 10S )

Procedure

  1. Preparation of E. coli strains
    1. Amplify the internal fragment (at least 500 bp) of the target gene from Anabaena chromosomal DNA.
    2. Clone the internal fragment into the multiple cloning sites (BglII-NotI-AgeI-SpeI-ApaI-SmaI/XmaI) of pZR606 (KmR/SpR) to produce the cargo plasmid [see details in (Chen et al., 2015)]. The map for multiple cloning sites in pZR606 is provided (Figure S2). The cargo plasmid must be transformed to E. coli NEB10β or E. coli DH10B.
    3. Grow E. coli HB101 harboring pRL443 (ApR) and pRL623 (CmR) in 2 ml LB broth with 100 µg/ml ampicillin and 25 µg/ml chloramphenicol overnight (~16 h) at 37 °C with 200 rpm shaking. Meanwhile, grow NEB10β bearing the cargo plasmid (KmR/SpR) in 2 ml LB broth with 50 µg/ml kanamycin overnight at 37 °C with 200 rpm shaking.
    4. Transfer 100 µl of the above overnight E. coli cultures into 5 ml fresh LB with appropriate antibiotics respectively; continue to grow for ~3 h (OD600 ~0.5).
    5. Harvest the 5 ml culture by centrifugation at 4,000 x g in Allegra X-15R for 10 min at 25 °C (room temperature).
    6. Wash the cell pellets three times with 1 ml LB to remove antibiotics completely, then add 200 μl LB to re-suspend the pellets, respectively.
    7. For mating experiment, mix 100 µl NEB10β bearing cargo plasmid with 100 µl HB101 harboring pRL443 and pRL623 and incubate at room temperature for 30 min (experimental group). For a negative control, mix 100 µl LB with 100 µl HB101 harboring pRL443 and pRL623 and incubate at room temperature for 30 min (control group).

  2. Preparation of Anabaena 7120 culture
    1. Grow Anabaena 7120 in 30 ml AA/8(N) medium (Allen & Arnon, 1955a, Allen & Arnon, 1955b) or BG11 medium (Rippka et al., 1979) for ca. 5 days until it reaches early exponential stage (OD700 ~0.5). The Anabaena 7120 is grown under continuous light illumination (ca. 50 μE/m2 s) at 30 °C and shaken at 120 rpm in a temperature controlled Innova-44R lighted incubator (New Brunswick Scientific) or in the Cyanobacteria Culture Room with continuous light illumination (ca. 50 μE/m2 s) at 30 °C and shaken at 120 rpm.
    2. Harvest the culture by centrifugation at 4,000 x g with Allegra X-15R for 10 min at 25 °C.
    3. Re-suspend the cell pellet with 2 ml AA/8(N) in a 25 ml glass flask, break filaments into an average 3-5 cell lengths (confirmed microscopically) by sonicating the cultures for 60-120 sec using water bath sonicator (Figure S3). Set up the sonicator under standard model and add water to the operating level line during the sonication process.
    4. Transfer the sonicated culture into a 15 ml centrifuge tube and centrifuge in Allegra X-15R at 4,000 x g for 10 min at 25 °C. Re-suspend the cell pellet with 1 ml AA/8(N), transfer the cells into a 1.5 ml Eppendorf tube, and then centrifuge at 6,000 x g for 1 min (Thermo Fisher Scientific, Micro17) at 25 °C. Re-suspend the pellet with 400 µl AA/8(N); then divide cells equally into two tubes.

  3. Conjugal transformation of a cargo plasmid into Anabaena 7120
    1. Mix 200 µl of Anabaena 7120 resuspension (step 11) with 200 µl of the mated E. coli mixture (experimental group, step 7), or the control mixture (control group, step 7), respectively, and incubate at room temperature for another 30 min.
    2. Spread the conjugal mixtures onto the autoclaved Immobilon-NC transfer membrane atop AA (N) agar containing 5% LB (v/v) without antibiotic and incubate at 30 °C, with continuous light illumination (ca. 50 μE/m2 s) for 24 h.
    3. Transfer the membrane onto AA (N) agar plate with 10 μg/ml spectinomycin (Sp10), and incubate under the same growth conditions until spectinomycin-resistant colonies gradually appear during 10-15 days as shown in Figure 1 (top and middle panels).


      Figure 1. Screen for the potential knockout mutants. The conjugal transfer mixture with the cargo plasmid was spread onto Immobilon-NC membrane atop AA (N) agar containing Sp10 for 0 (top panel) and 19 days (middle panel). The bottom panel shows the above individual colonies re-streaked onto a new AA (N) agar plate containing Sp10 for segregation. As a negative control, the conjugal transfer mixture without cargo plasmid (control) showed no individual colonies on AA (N) agar plate containing Sp10 (data not shown).

  4. Verification of the knockout mutants
    1. Re-streak at least ten potential knockout mutant colonies onto a fresh AA (N) agar plate containing Sp10, allowing complete segregation of mutant chromosomes (Figure 1, bottom panel) since Anabaena 7120 has multiple copies of chromosomes per cell (Hu et al., 2007).
    2. Screen for the positive knockout mutant colonies by colony PCR. Three primers were designed to verify the target gene knockout mutants. Targeted gene open reading frame (ORF) primer pair [forward primer (FP) and reverse primer (RP)] was used to verify the wild-type gene; while forward primer FP paired with a vector-specific primer ZR90 (AAGTTCTTCTCCTTTGCTAGC) (Chen et al., 2015) was used to distinguish the positive knockout mutants from WT. A standard PCR program was used with the exception that the chromosomal DNA template was prepared by preheating cyanobacterial cell suspension in 6 μl ddH2O at 95 °C for 10 min.
    3. Completely suspend PCR positive mutant colonies in 100 µl AA/8(N) medium to make serial dilutions (1x, 10x and 100x). Then re-streak each dilution onto AA (N) agar plate containing Sp10 for single colonies to separate E. coli contamination from Anabaena. These single Anabaena knockout mutant colonies were confirmed again with the colony PCR described above.
    4. Repeat steps 15 to 17 until no wild-type target gene was detected in the knockout mutant colony PCR. An example of colony PCR verification for the inactivated mutants of all4160 in Anabaena 7120 is shown in Figure 2.


      Figure 2. Colony PCR verification for the single crossover inactivated all4160 mutant in Anabaena 7120. The target gene specific F4160 + R4160 and F4160 + ZR90 primer pairs were used for PCR amplification from the knockout mutant colonies of all4160 (lanes 2-3 and 5-6, respectively) or from the WT 7120 (lanes 4 and 7) as controls. The PCR product sizes amplified with F4160 + R4160 and F4160 + ZR90 primer pairs are expected to be 1,056 bp (lane 7) and 910 bp (lanes 2-3), respectively. The sizes of DNA ladder (lane 1) are also indicated. Lanes 2 & 3 or lanes 5 & 6 were two independent knockout mutants. Lanes 5 & 6 show that the target gene was successfully knocked out, the mutants were completely segregated. F1460: ggatccatatgTACATGGCAACCAAAGTG, R4160: gtcgacATAAGCGCCACTATTTCTATTAAA (uppercase letters represent nucleotides corresponding to all4160).

  5. Complementation experiment
    1. A complementation experiment is required to confirm that the target gene, rather than a downstream gene, is responsible for the mutant phenotype, as in some cases, a knockout mutant’s phenotype might be due to a polar effect on the expression of a downstream gene. Complementation by a single gene was tested by sub-cloning the entirety of the target gene-coding region into a shuttle vector pZR670 (Xu et al., 2015) or pRL2833a (Wolk et al., 2007), which contains both the chloramphenicol (CmR) and the erythromycin (EmR) resistance genes. To construct the complementing plasmid, the coding sequence of the target gene is cloned into the multiple cloning sites (MCS) (NsiI/NdeI/AvrII/AatII/XhoI/AscI/NaeI/BamHI) of pZR670 (Figure 3), in-frame with the glnA start codon. For example, coding sequencing of alr4853 was in-frame cloned into NdeI-XhoI digested pZR670, producing pZR1617 (Xu et al., 2015).


      Figure 3. The multiple cloning sites of a complementing vector pZR670. PglnA: promoter region of glnA (alr2328), RBS: Ribosomal binding site, 5’-glnA: N-terminal portion (MTTPQEVLKRI) of Anabaena GlnA (glutamine synthetase), MCS (nucleotides highlighted in red): multiple cloning sites. The target gene-coding sequence (ORF) can be in-frame inserted between NdeI and one of AvrII/AatII/XhoI/AscI/NaeI/BamHI digested pZR670 (Xu et al., 2015).

    2. A similar approach described in Part C is used to transfer the complementing plasmid into the knockout mutant. E. coli and Anabaena mutant culture were prepared as described before (steps 3 to 11), except that the cargo plasmid was substituted by the complementing plasmid. Then dilute the mutant Anabaena culture by 5x, 10x and 100x with AA/8(N) in 1.5 ml centrifuge tubes. Spot 5 µl of the diluted cultures onto the Immobilon-NC membrane atop AA(N) agar containing 5% LB without antibiotic. Make 3 replicate spots for each sample. Air-dry the membrane for a few minutes; then spot 5 µl of the mated E. coli mixture atop the Anabaena spots. Air-dry for a few minutes, and incubate the plate under continuous light illumination (ca. 50 μE/m2 s) at 30 °C for 24 h.
    3. Transfer the membrane onto AA (N) agar plate containing both Sp10 and 10 μg/ml of erythromycin, and incubate under the same growth conditions. The Sp-Em-resistant colonies will grow in 10-15 days, similarly as seen in Figure 1.
    4. Then verify the Sp-Em-resistant colonies that contain the complementing plasmid by colony PCR with specific primers targeting the Em resistance gene.
    5. Examine the phenotype for at least three verified colonies to see if the complementing plasmid is capable of complementing the single crossover generated mutant. If the complemented mutant displays a restored wild-type phenotype, in which the target gene has been successfully inactivated by a single crossover approach, you can conclude that the target gene is responsible for the mutant’s phenotype, which hints the function of the target gene.
    6. If the complementing plasmid fails to restore the mutant’s phenotype to its wild-type’s phenotype in step 22, this indicates that the mutant’s phenotype might be caused by a polar effect on a downstream gene. No functional clue could be made for the target gene under this condition although it has been successfully inactivated.

Notes

  1. Anabaena sp. strain PCC 7120 can also be grown in BG11 medium (http://www-cyanosite.bio.purdue.edu/media/table/BG11.html) (Rippka et al., 1979).
  2. After transferring the membrane onto AA (N) agar plate with appropriate antibiotics, keep tracking the membrane color change. The wild-type Anabaena 7120 should be killed and the color of the membrane should gradually turn to yellow.
  3. Change the AA (N) agar plate with appropriate antibiotics every week to maintain effective antibiotics. If the membrane in the control plate (Anabaena 7120 + HB101 harboring pRL443 and pRL623) does not turn to yellow in one week, transfer all the membranes onto new AA(N) agar plate containing appropriate antibiotics immediately.

Recipes

  1. Allen and Arnon medium plus nitrate: AA/8(N)
    Anabaena 7120 is grown in a modified Allen-Arnon medium (Allen & Arnon, 1955a). AA liquid medium was diluted 8 times with sterilized distilled H2O to make AA/8. For making AA/8(N), a final concentration of 2.5 mM nitrate was added to AA/8 medium from KNO3/NaNO3 stock solution. For preparation of AA(N) agar plates, a final concentration of 2.5 mM nitrate and 1% agar were added to AA medium.
    1. AA medium preparation
      Solution A 25.0 ml
      Solution B 6.25 ml
      Agar (if needed) 10.0 g
      Distilled water 969.0 ml
      Aliquot 200 ml into five bottles, and then autoclave them at 121 °C for 20 min
    2. Solution A (A & A minus phosphate stock solution)
      4% MgSO4·7H2O 500.0 ml (final concentration 1%, m/m)
      1.2% CaCl2·2H2O 500.0 ml (final concentration 0.3%, m/m)
      3.8% NaCl 500.0 ml (final concentration 0.95%, m/m)
      Microelements stock solution 500.0 ml (final concentration of microelements in solution A is four times diluted microelements stock solution) 
    3. Solution B (K2HPO4 stock solution)
      K2HPO4 21.40 g
      Distilled water 500.0 ml
    4. KNO3/NaNO3 stock solution (500 mM)
      KNO3 25.276 g
      NaNO3 21.249 g
      Add distilled H2O to a final volume of 500 ml
      Stock solution is autoclaved and stored at 4 °C
    5. Microelements stock solution
      A & A FeEDTA solution 160.0 ml
      MnCl2·4H2O 360 mg
      Na2MoO4·2H2O 61.1 mg
      ZnSO4·7H2O 44.0 mg
      CuSO4·5H2O 15.8 mg
      H3BO3 572.0 mg
      NH4VO3 4.6 mg
      CoCl2·6H2O 8.0 mg
      Distilled water 1,090.0 ml
    6. A & A FeEDTA stock solution
      Dissolve 5.2 g KOH in 186 ml distilled water, add 20.4 g Na2EDTA·2H2O
      Dissolve 13.7 g FeSO4·7H2O in 364 ml distilled water
      Mix the above two solutions, then bubbling millipore-filtered air through solution until color changes. The final pH of FeEDTA solution is approximately 7.5.
    Note: All stock solutions are recommended to store at 4 °C.

Acknowledgments

The protocol is based on the publications “Conjugal transfer of DNA to cyanobacteria” (Elhai and Wolk, 1988); “Simultaneous gene inactivation and promoter reporting in cyanobacteria” (Chen et al., 2015), and “Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120” (Xu et al., 2015). The authors would like to thank Jaimie Gibbons for her critical reading of the manuscript. This work was partially supported by the NSF, Energy for Sustainability Grant CBET1133951 (to R. Z.), and by the USDA-NIFA grant 11665597 (to R. Z.).

References

  1. Allen, M. B. and Arnon, D. I. (1955). Studies on nitrogen-fixing blue-green algae. I. growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30(4): 366-372.
  2. Chen, K., Xu, X., Gu, L., Hildreth, M. and Zhou, R. (2015). Simultaneous gene inactivation and promoter reporting in cyanobacteria. Appl Microbiol Biotechnol 99(4): 1779-1793.
  3. Elhai, J., Vepritskiy, A., Muro-Pastor, A. M., Flores, E. and Wolk, C. P. (1997). Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179(6): 1998-2005.
  4. Elhai, J. and Wolk, C. P. (1988). Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167: 747-754.
  5. Golden, J. W. and Yoon, H. S. (2003). Heterocyst development in Anabaena. Curr Opin Microbiol 6(6): 557-563.
  6. Halfmann, C., Gu, L. and Zhou, R. (2014). Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O. Green Chemistry 16: 3175-3185.
  7. Hu, B., Yang, G., Zhao, W., Zhang, Y. and Zhao, J. (2007). MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 63(6): 1640-1652.
  8. Land, M., Hauser, L., Jun, S. R., Nookaew, I., Leuze, M. R., Ahn, T. H., Karpinets, T., Lund, O., Kora, G., Wassenaar, T., Poudel, S. and Ussery, D. W. (2015). Insights from 20 years of bacterial genome sequencing. Funct Integr Genomics 15(2): 141-161.
  9. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111: 1-61.
  10. Wolk, C. P., Fan, Q., Zhou, R., Huang, G., Lechno-Yossef, S., Kuritz, T. and Wojciuch, E. (2007). Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120. Arch Microbiol 188(6): 551-563.
  11. Xu, X., Elhai, J. and Wolk, C. (2008). Transcriptional and developmental responses by Anabaena to deprivation of fixed nitrogen. Caister Academic Press, Norfolk, United Kingdom.
  12. Xu, X., Gu, L., He, P. and Zhou, R. (2015). Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120. Microbiology 161(6): 1219-1230.

简介

菌株PCC 7120长期充当用于研究N 2 - 固定,光合作用和各种植物类型代谢途径和生物燃料生产以及细胞分化的模式生物体(Xu等人,/em>。,2008,Halfmann等人,2014,Golden and Yoon,2003)。由于目前可获得超过30,000个测序的细菌基因组(Land等人,2015),特异性基因失活和相应突变体表型的分析已成为阐明靶基因功能的有力工具。在这里,我们描述了灭活anabaena sp中的靶基因的方案。 PCC 7120使用单交叉方法。该方法仅需要将靶基因的内部片段一步克隆到整合载体中以产生货物质粒。在货物质粒和鱼腥藻染色体之间的单次交换(同源重组)时,内源靶基因通过产生3'-和5'-缺失的片段而被破坏。该基因失活方案基于整合载体pZR606(Chen等人,2015),其可以广泛应用于其他蓝细菌物种以及其他原核生物中的基因失活。

关键字:细菌的遗传工具, 遗传工程, 整合载体的方法, 单交叉法, 合成的蓝藻

材料和试剂

  1. 培养皿(Fisher Scientific,目录号:FB0875713)
  2. 玻璃烧瓶(Fisher Scientific,目录号:FB-500-50)
  3. 1.5ml离心管(Fisher Scientific,目录号:02-682-550)
  4. 15ml锥形离心管(Fisher Scientific,目录号:05-527-90)
  5. 50ml锥形离心管(Fisher Scientific,目录号:12-565-270)
  6. 应变PCC 7120(以下称为"Anabaena 7120")
  7. 大肠杆菌(大肠杆菌)NEB10β[Δ(ara-leu)7697 araD139 fhuAΔlacX74 galK16 galE15 e14-Φ80< (mrr-hsdRMS-mcrBC dph),其中所述mrr-hsdRMS-mcrBC d lacZ Δm15recA1 relA1 endA1 nupG rpsL(StrR) em>); New England BioLabs,目录号:C3019H]
  8. E。大肠杆菌HB101 [F - mcrB mrr ems hsd S20(r subB - m < B6, ara -14, pro em> A2, lac Y1, K2, xyl -5, mtl -1, rps L20(Sm R ) gln V44λ - ; Promega,目录号:L2015]
  9. 结合质粒pRL443和辅助质粒pRL623(Elhai等人,1997)
  10. pZR606,一种用于 anabaena 7120的整合载体(Chen 。,2015)。 pZR606可根据要求提供(GenBank,目录号:KJ500179.1)
  11. pZR670(可根据要求提供),Anabaena中的复制型载体 7120(Xu et al。,205)[提供pZR670的图谱(图S1 )。]
  12. LB肉汤(Sigma-Aldrich,目录号:L3522-1KG)
  13. LB琼脂,用于生长E。大肠杆菌(MP,目录号:100262)
    注意:这个品牌的琼脂不适用于蓝细菌。
  14. Agar,生长Anabaena 7120和其他蓝细菌(Fisher Scientific,目录号:A360-500)所需的。
  15. Immobilon-NC转移膜(Millipore,目录号:HATF08550)
  16. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518-25G)
  17. 氯霉素(Fisher Scientific,目录号:BP904-100)
  18. 红霉素(Sigma-Aldrich,目录号:E6376-25G)
  19. 硫酸卡那霉素(Sigma-Aldrich,目录号:K4000-25G)
  20. 壮观霉素二盐酸盐五水合物(Sigma-Aldrich,目录号:S9007-25G)
  21. MgSO 4·7H 2 O(MP,目录号:194833)
  22. CaCl 2 2·2H 2 O(Fisher Scientific,目录号:BP510-500)
  23. NaCl(Fisher Scientific,目录号:S271-1)
  24. (Fisher Scientific,目录号:BP363-500)
  25. KNO 3(Fisher Scientific,目录号:BP368-500)
  26. NaNO 3(Fisher Scientific,目录号:BP360-500)
  27. MnCl 2·4H 2 O(Fisher Scientific,目录号:M87-100)
  28. Na 2 Mo 4+ 2H 2 O(99%纯度)(Acros Organics,目录号:206371000)
  29. ZnSO 4·7H 2 O(Fisher Scientific,目录号:Z76-500)
  30. CuSO 4·5H 2 O(Fisher Scientific,目录号:BP346-500)

  31. (Fisher Scientific,目录号:BP168-500)

  32. (Acros Organics,目录号:194910500)。
  33. CoCl 2 6H·6H 2 O(Fisher Scientific,目录号:C371-100)
  34. KOH(Fisher Scientific,目录号:P250-500)
  35. Na 2 EDTA·2H 2 O(Fisher Scientific,目录号:BP120-500)
  36. FeSO 4 7HH 2 O(Fisher Scientific,目录号:I146-500)
  37. Allen和Arnon培养基加硝酸盐:AA/8(N)(参见配方)

设备

  1. 离心机(Beckman,型号:Allegra X-15R; Thermo Fisher Scientific,Sorvall TM supec TM 型号:Micro17)
  2. 将配备有连续荧光照明(约50-100μE/m 2 s/s)(Eppendorf,New Brunswick sup>,型号:44R)
  3. 配备有数字温度控制系统和Sylvania荧光灯泡(F40CWX,40W/4100K,T12)的蓝细菌培养室(恒温在30℃)
    注意:蓝藻培养室中的浅色架子能够提供连续的光照(约50-150μE/m 2 ·)。
  4. ESCO层流柜(ESCO,Clean BenchAirstream 型号:AHC-4B2)
  5. 水浴超声器(Bransonic超声波清洗机,型号:1510R-MT)
  6. 显微镜,奥林巴斯立式复合显微镜(Olympus,型号:AX70; BX53)
  7. PCR热循环仪(Bio-Rad,Touch TM ,型号:C1000)
  8. UV-Vis分光光度计(Thermo Fisher Scientific,型号:GeneSyS 10S)

程序

  1. E的制备。大肠杆菌菌株
    1. 扩增来自鱼腥藻染色体DNA的靶基因的内部片段(至少500bp)。
    2. 将内部片段克隆到多个克隆位点( Bg lII- 不是 I- p RR606(Km /Ap I-Sma I/ Xma sup>)以产生货物质粒[参见(Chen等人,2015)中的细节]。提供了pZR606中多克隆位点的图谱(图S2 )。货物质粒必须转化为大肠杆菌NEB10β或大肠杆菌DH10B。
    3. 成长。在含有100μg/ml氨苄青霉素和25μg/ml氯霉素的2ml LB肉汤中培养含有pRL443(Ap )和pRL623(Cm R)的大肠杆菌HB101 (?16h),在37℃,200rpm振荡。同时,在具有50μg/ml卡那霉素的2ml LB肉汤中在37℃下以200rpm振荡培养携带货物质粒(Km /Sp R )的NEB10β过夜。
    4. 转移100微升的上述过夜E。大肠杆菌培养物分别加入含有合适抗生素的5ml新鲜LB;继续生长?3小时(OD <600
    5. 通过在25℃(室温)下在Allegra X-15R中以4,000×g离心10分钟收获5ml培养物。
    6. 用1ml LB洗涤细胞沉淀三次,以完全除去抗生素,然后加入200μlLB分别重新悬浮沉淀。
    7. 对于交配实验,100 100 NEB106携带货物质粒与100微升含有pRL443和pRL623的HB101,并在室温下孵育30分钟(实验组)。对于阴性对照,将100μlLB与含有pRL443和pRL623的100μlHB101混合,并在室温下孵育30分钟(对照组)。

  2. 准备 7120文化
    1. 在30ml AA/8(N)培养基(Allen& Arnon,1955a,Allen& Arnon,1955b)或BG11培养基(Rippka等人)中培养Anabaena 7120。 ,1979)。 5天直到它达到早期指数期(OD <700)。在30℃下在连续光照(约50μE/m 2 s)下生长并在温度控制的Innova-44R中以120rpm振荡的 (New Brunswick Scientific)或在具有连续光照明(约50μE/m 2秒)的蓝藻培养室中,在30℃下以120rpm振荡。
    2. 通过在25℃下用Allegra X-15R在4,000×g离心10分钟收获培养物。
    3. 用2ml AA/8(N)在25ml玻璃烧瓶中重新悬浮细胞沉淀,通过超声处理培养物60-120秒,使用水浴超声仪图S3 )。在标准模型下设置超声波仪,并在超声处理过程中向操作水平线添加水
    4. 将超声处理的培养物转移到15ml离心管中并在4℃下在Allegra X-15R中在25℃下离心10分钟。用1ml AA/8(N)重悬细胞沉淀,将细胞转移到1.5ml Eppendorf管中,然后在6,000×g离心1分钟(Thermo Fisher Scientific,Micro17)1分钟25℃。用400μlAA/8(N)重悬浮沉淀;然后将细胞平均分成两个管
  3. 将货物质粒共轭转化为 7120
    1. 混合200μl的 7120重悬浮(步骤11)与200μl的交配的E。大肠杆菌混合物(实验组,步骤7)或对照混合物(对照组,步骤7),并在室温下再温育30分钟。
    2. 将共轭混合物铺展到含有5%LB(v/v)的没有抗生素的AA(N)琼脂上的高压灭菌的Immobilon-NC转移膜上,并在30℃下,连续光照(约50μE/2 ?s)培养24小时。
    3. 将膜转移到具有10μg/ml壮观霉素(Sp10)的AA(N)琼脂板上,并在相同的生长条件下孵育直至壮观霉素抗性菌落在10-15天内逐渐出现,如图1所示(顶部和中间图) 。


      图1.潜在敲除突变体的筛选将具有货物质粒的共轭转移混合物涂布在含有Sp10的AA(N)琼脂顶上的Immobilon-NC膜上(< em>)和19天(中间面板)。底部图显示将上述单个菌落重划线到含有用于分离的Sp10的新AA(N)琼脂平板上。作为阴性对照,无货物质粒的共轭转移混合物(对照)在含有Sp10的AA(N)琼脂平板上没有显示单个菌落(数据未显示)。

  4. 敲除突变体的验证
    1. 将至少10个潜在敲除突变体菌落重新连接到含有Sp10的新鲜AA(N)琼脂平板上,使得突变体染色体完全分离(图1,底部图),因为鱼??腥藻7120具有多个染色体拷贝每个细胞(Hu等人,2007)。
    2. 通过菌落PCR筛选阳性敲除突变体菌落。设计三个引物以验证靶基因敲除突变体。使用靶向基因开放阅读框(ORF)引物对[正向引物(FP)和反向引物(RP)]来验证野生型基因;而使用与载体特异性引物ZR90(AAGTTCTTCTCCTTTGCTAGC)(Chen等人,2015)配对的正向引物FP来区分来自WT的阳性敲除突变体。使用标准PCR程序,除了通过在6μlddH 2 O中在95℃下预热蓝细菌细胞悬浮液10分钟来制备染色体DNA模板。
    3. 完全悬浮PCR阳性突变体菌落在100微升AA/8(N)培养基,使系列稀释(1x,10x和100x)。然后将每个稀释液重新连接到含有Sp10的AA(N)琼脂平板上用于单菌落以分离E。大麻污染 。使用上述菌落PCR再次确认这些单个鱼腥藻敲除突变体菌落。
    4. 重复步骤15至17,直到在敲除突变菌落PCR中没有检测到野生型靶基因。图2中显示了 anabaena 7120中 all4160 的灭活突变体的菌落PCR验证的实例。


      图2.在鱼腥藻7120中单个交换灭活的 all4160 突变体的菌落PCR验证。目标基因特异性F 4160 使用来自all4160的敲除突变菌落的PCR扩增(泳道2-3和泳道2-3)用于PCR扩增,其中使用+ R sub 4160和F sub 4160 + ZR90引物对5-6)或来自WT 7120(泳道4和7)作为对照。用F 4160+ R 4160和F 4160+ + ZR90引物对扩增的PCR产物大小预期为1,056bp(泳道7)和(泳道2-3)。还显示了DNA梯形(泳道1)的大小。泳道2& 3或泳道5& 6是两个独立的敲除突变体。泳道5&图6显示靶基因被成功敲除,突变体完全分离。 F 1460 :ggatccatatgTACATGGCAACCAAAGTG,R :gtcgacATAAGCGCCACTATTTCTATTAAA(大写字母表示对应于 all4160 的核苷酸)。

  5. 互补实验
    1. 需要互补实验以证实靶基因而不是下游基因负责突变表型,因为在一些情况下,敲除突变体表型可能是由于对下游基因的表达的极性效应。通过将整个靶基因编码区亚克隆到穿梭载体pZR670(Xu等人,2015)或pRL2833a(Wolk等人,2010)中来测试单个基因的互补,/2007),其含有氯霉素(C max)和红霉素(Em sup R)抗性基因。为了构建互补质粒,将靶基因的编码序列克隆到多克隆位点(MCS)(NsiI/IeI/Nde I/em/I/em/I/em/II/I am/I am/I am I/(图3),与glnA起始密码子符合读框。例如,将alr4853的编码测序框内克隆到经消化的pZR670的Nde I-Xho I中,产生pZR1617 al 。,2015)。


      图3.互补载体pZR670的多克隆位点。 p glnA :glnA的启动子区域( alr2328 ),RBS:核糖体结合位点,5'-glnA:鱼腥藻GlnA(谷氨酰胺合成酶)的N-末端部分(MTTPQEVLKRI),MCS(以红色突出显示的核苷酸):多克隆位点。靶基因编码序列(ORF)可以框内插入Nde I I和Avr II/em/Aat II/em之一> Xho I/ Asc I/ Nae I/ BamH I digested pZR670(Xu 。,2015)。

    2. 部分C中描述的类似方法用于将互补质粒转移到敲除突变体中。 E。 (步骤3至11)制备大肠杆菌和洋葱突变体培养物,除了货物质粒被互补质粒代替。然后用1.5ml离心管中的AA/8(N)稀释突变鱼腥藻培养物5x,10x和100x。将5μl稀释的培养物点样在含有5%不含抗生素的LB的AA(N)琼脂上的Immobilon-NC膜上。每个样品做3个重复点。空气干燥膜几分钟;然后点5μl的交配的E。大肠杆菌混合物在 Anabaena 点上。空气干燥几分钟,并在30℃下在连续光照(约50μE/m 2秒)下孵育该板24小时。
    3. 将膜转移到含有Sp10和10μg/ml红霉素的AA(N)琼脂板上,并在相同的生长条件下孵育。 Sp-Em-抗性菌落将在10-15天内生长,类似于图1所示
    4. 然后通过用针对Em抗性基因的特异性引物进行菌落PCR验证含有互补质粒的Sp-Em-抗性菌落。
    5. 检查至少三个验证的菌落的表型,看看互补质粒是否能够补充单交换产生的突变体。如果互补的突变体显示恢复的野生型表型,其中靶基因已经通过单一交叉方法成功灭活,您可以得出结论,目标基因负责突变体的表型,暗示目标基因的功能。
    6. 如果互补质粒未能在步骤22中将突变体表型恢复为其野生型表型,则这表明突变体表型可能由对下游基因的极性效应引起。在这种条件下,尽管其已经成功灭活,但是对于靶基因没有功能线索。

笔记

  1. 菌株PCC 7120也可以在BG11培养基中生长( http://www-cyanosite.bio.purdue.edu/media/table/BG11.html )(Rippka等人。,1979)。
  2. 将膜转移到具有适当抗生素的AA(N)琼脂板上,保持跟踪膜颜色变化。野生型 anabaena 7120应杀死,膜的颜色应逐渐变为黄色。
  3. 每周更换适当的抗生素的AA(N)琼脂板,以维持有效的抗生素。如果对照板(含有pRL443和pRL623的Anabaena 7120 + HB101)的膜在一周内没有变成黄色,则立即将所有膜转移到含有合适抗生素的新AA(N)琼脂板上。

食谱

  1. Allen和Arnon培养基加硝酸盐:AA/8(N)
    7120在改良的Allen-Arnon培养基(Allen& Arnon,1955a)中生长。 AA液体培养基用无菌蒸馏H 2 O稀释8倍以制备AA/8。为了制备AA/8(N),将最终浓度为2.5mM的硝酸盐加入到来自KNO 3/NaNO 3储备溶液的AA/8培养基中。为了制备AA(N)琼脂平板,将2.5mM硝酸盐和1%琼脂的终浓度加入AA培养基中。
    1. AA介质制剂
      解决方案A  25.0 ml
      溶液B 6.25ml
      琼脂(如果需要)10.0克
      蒸馏水969.0 ml
      将200ml分装到五个瓶中,然后在121℃下高压灭菌20分钟
    2. 溶液A(A& A减磷酸盐储备溶液)
      4%MgSO 4·7H 2 O 500.00ml(最终浓度1%,m/m)
      1.2%CaCl 2·2H 2 O 500.0ml(终浓度0.3%,m/m)
      3.8%NaCl 500.0ml(终浓度0.95%,m/m) 微量元素储备溶液500.0ml(溶液A中微量元素的最终浓度为四倍稀释的微量元素储备溶液)
    3. 溶液B(K 2 HPO 4储备溶液)
      HPO 21.40 g
      蒸馏水500.0 ml
    4. KNO 3/NaNO 3储备溶液(500mM)
      KNO 3 25.276克
      NaNO <3> 21.249克
      加入蒸馏的H 2 O至最终体积为500ml
      将原液高压灭菌并在4℃下贮存
    5. 微量元素储液
      A& FeEDTA溶液160.0ml
      MnCl 2 2·4H 2 O 360mg/dm 2 ZnSO 4·7H 2 O 44.0mg
      CuSO 4·5H 2 O 15.8mg
      H sub 3 3 BO 3 572.0mg

      CoCl 2 2·6H 2 O 8.0mg/dm 2 蒸馏水1,090.0 ml
    6. A& FeEDTA储备溶液
      将5.2g KOH溶解在186ml蒸馏水中,加入20.4g Na 2 EDTA 2·2H 2 O·h / 在364ml蒸馏水中溶解13.7g FeSO 4·7H 2 O。 混合以上两种溶液,然后鼓泡微孔过滤的空气通过溶液,直到颜色变化。 FeEDTA溶液的最终pH为约7.5。

致谢

该协议基于出版物"DNA到蓝细菌的共轭转移"(Elhai和Wolk,1988); "在蓝细菌中的同时基因失活和启动子报告"(Chen等人,2015),和"Characterization of five putative aspartate aminotransferase genes in the N sub 2 -fixing heterocystous cyanobacterium 菌株PCC 7120"(Xu等人,2015)。作者感谢Jaimie Gibbons对于手稿的批判性阅读。这项工作得到NSF,可持续发展能源补助金CBET1133951(R.Z.),以及USDA-NIFA补助金11665597(R.Z.)的部分支持。

参考文献

  1. Allen,MB和Arnon,DI(1955)。  研究对固氮蓝绿藻。 I.生长和氮固定通过 Lemm 植物生理 30(4):366-372。
  2. Chen,K.,Xu,X.,Gu,L.,Hildreth,M.和Zhou,R。(2015)。  在蓝细菌中同时基因失活和启动子报告。 Appl Microbiol Biotechnol 99(4):1779-1793。 />
  3. Elhai,J.,Vepritskiy,A.,Muro-Pastor,AM,Flores,E.and Wolk,CP(1997)。  通过三种限制性活性的鱼腥藻属物种减少共轭转移效率。菌株PCC 7120. J Bacteriol 179(6):1998-2005。
  4. Elhai,J。和Wolk,CP(1988)。  DNA与蓝细菌的共轭转移。方法Enzymol 167:747-754。
  5. Golden,JW和Yoon,HS(2003)。  Heterocyst 6(6):557-563。
  6. Halfmann,C.,Gu,L.和Zhou,R。(2014)。  工程用蓝藻,用于从CO 2和H 2 O生产环状烃燃料。 em> Green Chemistry 16:3175-3185。
  7. Hu,B.,Yang,G.,Zhao,W.,Zhang,Y.and Zhao,J.(2007)。  20分钟的洞察年的细菌基因组测序。 Funct Integr Genomics 15(2):141-161。
  8. Rippka,R.,Deruelles,J.,Waterbury,JB,Herdman,M.and Stanier,RY(1979)。  蓝藻菌纯培养物的通用分配,菌株历史和性质。微生物学111:1 -61。
  9. Wolk,CP,Fan,Q.,Zhou,R.,Huang,G.,Lechno-Yossef,S.,Kuritz,T.and Wojciuch,E。(2007)。  用于互补蓝细菌鱼腥藻中突变的配对克隆载体。菌株PCC 7120. Arch Microbiol 188(6):551-563。
  10. 徐,X.,Elhai,J.和沃尔克,C.(2008)&NBSP; <一类="KE-的insertFile的"href ="https://books.google.com.tw/books?hl=zh- CN&LR =&ID = xgMahO1BXrQC与OI = FND与PG = RA1-PA1992和DQ =转录+和+发展+响应+通过+鱼腥+固定+中+剥夺+ +氮和OTS = m7epeZ6Qps与SIG = PSVBpgvYK_6wCjf3RRPme9r8N0c&redir_esc = Y#v = onepage&q =转录%20于是%20developmental%通过20responses%20by%20Anabaena%20to%20deprivation%20of%20fixed%20nitrogen&F =假"目标="_空白">转录和发育反应的鱼腥的固定氮剥夺。的 Caister 学术出版社,Norfolk,英国
  11. 徐,X.,谷,湖,河,P.,周,R.(2015)&NBSP; <一类="KE-的insertFile的"href ="http://www.ncbi.nlm.nih.gov /考研/25808172"目标="_空白">在N2-固定heterocystous蓝藻的鱼腥的SP 5公认的天冬氨酸氨基转移酶基因的表征。菌株PCC 7120. 微生物学 161(6):1219-1230。
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引用:Chen, K., Zhu, H., Gu, L., Tian, S. and Zhou, R. (2016). Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120. Bio-protocol 6(15): e1890. DOI: 10.21769/BioProtoc.1890.
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