Cultivarium POSSUM Toolkit
(Kit #
1000000234
)
Depositing Lab: Nili Ostrov, Charlie Gilbert, Cultivarium Tools
The POSSUM Toolkit (Plasmid Origins and Selectable markerS for Undomesticated Microbes) is a Golden Gate genetic toolkit developed to help researchers identify functional plasmids and establish genetic tractability in non-model microbes. The kit includes plasmid origins of replication and selection markers for a variety of bacterial and fungal species and other genetic parts for engineering non-model microbes.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria Gilbert C, Brumwell SL, Crits-Christoph A, Kang SC, Martin-Moldes Z, Alsharif W, Esmurria A, Nguyen M-A, Lee HH, Ostrov N. bioRxiv 2023.05.19.541510. doi: 10.1101/2023.05.19.541510. Article (Link opens in a new window).
Description
Establishing plasmid transformation is a fundamental step in the genetic manipulation of microorganisms, underpinning heterologous protein expression, genome engineering, gene editing, and other applications. Screening for functional origin of replication (ORI) sequences and selection markers is essential to this process, but there is a lack of standardized, scalable resources to achieve this in non-model microbes. To make this process simpler, cheaper, faster, and more accessible to researchers, we have developed the Plasmid Origins and Selectable markerS for Undomesticated Microbes (POSSUM) Toolkit. The kit provides 111 Level 0 plasmids, including ORIs and selection markers for a range of bacterial and fungal species, and 22 destination vectors, as well as all the components required to assemble them together for multiplexed plasmid screening in non-model microbes. These parts are compatible with dozens of other Golden Gate toolkits.
- Figure 1: Overview of the POSSUM Toolkit (left). Illustration of the Loop Assembly method employed by the POSSUM Toolkit (right). Alternating rounds of BsaI and SapI Golden Gate assemblies combine up to four parts at a time into destination vectors. Assembly levels alternate between Odd and Even destination vectors. oriT: conjugation origin of transfer; RBS: ribosome binding site; CDS: coding sequence; NGS: next-generation sequencing. Possum image generated using MidJourney.
Kit Documentation
- Level 0 Plasmid Maps (GB, 623 KB)
- Level 1 Plasmid Maps (GB, 184 KB)
- Level 2 Plasmid Maps (GB, 279 KB)
- Destination Vector Plasmid Maps (GB, 152 KB)
- Other Plasmid Maps (GB, 31 KB)
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The Cultivarium POSSUM Toolkit was a gift from Nili Ostrov, Charlie Gilbert, and Cultivarium Tools (Addgene kit #1000000234).”
For your Reference section:
A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria Gilbert C, Brumwell SL, Crits-Christoph A, Kang SC, Martin-Moldes Z, Alsharif W, Esmurria A, Nguyen M-A, Lee HH, Ostrov N. bioRxiv 2023.05.19.541510. doi: 10.1101/2023.05.19.541510. Article (Link opens in a new window).
Cultivarium POSSUM Toolkit - #1000000234
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column links to its kit plate map on the right, as well as lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Chloramphenicol | |
| Chloramphenicol and Gentamicin | |
| Kanamycin | |
| Ampicillin | |
| Chloramphenicol and Kanamycin | |
| Chloramphenicol and Tetracycline | |
| Chloramphenicol and Bleocin (Zeocin) | |
| Chloramphenicol and Erythromycin | |
| Spectinomycin | |
| Chloramphenicol and Ampicillin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| Plate 1 / A / 1 | pGL0_1 [RK2 ORI] | Chloramphenicol |
| Plate 1 / A / 2 | pGL0_2 [pBBR1 ORI] | Chloramphenicol |
| Plate 1 / A / 3 | pGL0_3 [RSF1010 ORI] | Chloramphenicol |
| Plate 1 / A / 4 | pGL0_4 [pIJ101 ORI] | Chloramphenicol |
| Plate 1 / A / 5 | pGL0_5 [pAL5000 ORI] | Chloramphenicol |
| Plate 1 / A / 6 | pGL0_7 [pSa ORI] | Chloramphenicol |
| Plate 1 / A / 7 | pGL0_9 [pUB110 ORI] | Chloramphenicol |
| Plate 1 / A / 8 | pGL0_11 [pWKS1 ORI] | Chloramphenicol |
| Plate 1 / A / 9 | pGL0_12 [pAMβ1 ORI] | Chloramphenicol |
| Plate 1 / A / 10 | pGL0_13 [pIP501 ORI] | Chloramphenicol |
| Plate 1 / A / 11 | pGL0_15 [CEN6/ARSH4 ORI] | Chloramphenicol |
| Plate 1 / A / 12 | pGL0_16 [2μ ORI] | Chloramphenicol |
| Plate 1 / B / 1 | pGLEL_6 [EF] | Chloramphenicol |
| Plate 1 / B / 2 | pGL0_18 [AMA1 long ORI] | Chloramphenicol |
| Plate 1 / B / 3 | pGL0_19 [panARS ORI] | Chloramphenicol |
| Plate 1 / B / 4 | pGL0_20 [panARS-opt ORI] | Chloramphenicol |
| Plate 1 / B / 5 | pGL0_21 [UARS1 ORI] | Chloramphenicol |
| Plate 1 / B / 6 | pGL0_22 [pSG5 ORI] | Chloramphenicol |
| Plate 1 / B / 7 | pGL0_23 [pSC101ts ORI] | Chloramphenicol |
| Plate 1 / B / 8 | pGL0_24 [pBBR1-UP ORI] | Chloramphenicol |
| Plate 1 / B / 9 | pGL0_25 [P3 promoter] | Chloramphenicol |
| Plate 1 / B / 10 | pGL0_26 [bla pBR322] | Chloramphenicol |
| Plate 1 / B / 11 | pGL0_27 [Tfd gene VIII] | Chloramphenicol |
| Plate 1 / B / 12 | pGL0_28 [Paph(3')-I EZ-Tn5] | Chloramphenicol |
| Plate 1 / C / 1 | pGL0_29 [aph(3')-I EZ-Tn5] | Chloramphenicol |
| Plate 1 / C / 2 | pGL0_30 [PaphA-3 ] | Chloramphenicol |
| Plate 1 / C / 3 | pGL0_31 [aphA-3] | Chloramphenicol |
| Plate 1 / C / 4 | pGL0_32 [Pcat] | Chloramphenicol |
| Plate 1 / C / 5 | pGL0_33 [cat] | Chloramphenicol |
| Plate 1 / C / 6 | pGL0_34 [PaadA] | Chloramphenicol |
| Plate 1 / C / 7 | pGL0_35 [aadA] | Chloramphenicol |
| Plate 1 / C / 8 | pGL0_36 [PtetA pSC101] | Chloramphenicol |
| Plate 1 / C / 9 | pGL0_37 [tetA pSC101] | Chloramphenicol |
| Plate 1 / C / 10 | pGL0_38 [Paac(3)-Ia] | Chloramphenicol |
| Plate 1 / C / 11 | pGL0_39 [aac(3)-Ia] | Chloramphenicol |
| Plate 1 / C / 12 | pGL0_40 [dfrA1] | Chloramphenicol |
| Plate 1 / D / 1 | pGL0_41 [Paac3-IV] | Chloramphenicol |
| Plate 1 / D / 2 | pGL0_42 [aac3-IV] | Chloramphenicol |
| Plate 1 / D / 3 | pGL0_43 [tsr] | Chloramphenicol |
| Plate 1 / D / 4 | pGL0_44 [hph] | Chloramphenicol |
| Plate 1 / D / 5 | pGL0_45 [nat1] | Chloramphenicol |
| Plate 1 / D / 6 | pGL0_46 [nptII] | Chloramphenicol |
| Plate 1 / D / 7 | pGL0_47 [bar] | Chloramphenicol |
| Plate 1 / D / 8 | pGL0_48 [ble] | Chloramphenicol |
| Plate 1 / D / 9 | pGL0_49 [tk101] | Chloramphenicol |
| Plate 1 / D / 10 | pGL0_50 [catE] | Chloramphenicol |
| Plate 1 / D / 11 | pGL0_51 [PcatA] | Chloramphenicol |
| Plate 1 / D / 12 | pGL0_52 [catA] | Chloramphenicol |
| Plate 1 / E / 1 | pGL0_53 [TcatA] | Chloramphenicol |
| Plate 1 / E / 2 | pGL0_54 [PermC] | Chloramphenicol |
| Plate 1 / E / 3 | pGL0_55 [ermC] | Chloramphenicol |
| Plate 1 / E / 4 | pGL0_56 [TermC] | Chloramphenicol |
| Plate 1 / E / 5 | pGL0_57 [PtetL] | Chloramphenicol |
| Plate 1 / E / 6 | pGL0_58 [tetL] | Chloramphenicol |
| Plate 1 / E / 7 | pGL0_59 [TtetL] | Chloramphenicol |
| Plate 1 / E / 8 | pGL0_60 [sfGFP] | Chloramphenicol |
| Plate 1 / E / 9 | pGL0_62 [sfYFP] | Chloramphenicol |
| Plate 1 / E / 10 | pGL0_64 [mScarlet] | Chloramphenicol |
| Plate 1 / E / 11 | pGL0_65 [StayGold] | Chloramphenicol |
| Plate 1 / E / 12 | pGL0_76 [ORI_BC_1] | Chloramphenicol |
| Plate 1 / F / 1 | pGL0_77 [ORI_BC_2] | Chloramphenicol |
| Plate 1 / F / 2 | pGL0_78 [ORI_BC_3] | Chloramphenicol |
| Plate 1 / F / 3 | pGL0_79 [ORI_BC_4] | Chloramphenicol |
| Plate 1 / F / 4 | pGL0_80 [ORI_BC_5] | Chloramphenicol |
| Plate 1 / F / 5 | pGL0_81 [ORI_BC_6] | Chloramphenicol |
| Plate 1 / F / 6 | pGL0_82 [ORI_BC_7] | Chloramphenicol |
| Plate 1 / F / 7 | pGL0_83 [ORI_BC_8] | Chloramphenicol |
| Plate 1 / F / 8 | pGL0_84 [ORI_BC_9] | Chloramphenicol |
| Plate 1 / F / 9 | pGL0_85 [ORI_BC_10] | Chloramphenicol |
| Plate 1 / F / 10 | pGL0_86 [ORI_BC_11] | Chloramphenicol |
| Plate 1 / F / 11 | pGL0_87 [ORI_BC_12] | Chloramphenicol |
| Plate 1 / F / 12 | pGL0_88 [ORI_BC_13] | Chloramphenicol |
| Plate 1 / G / 1 | pGL0_89 [ORI_BC_14] | Chloramphenicol |
| Plate 1 / G / 2 | pGL0_90 [ORI_BC_15] | Chloramphenicol |
| Plate 1 / G / 3 | pGL0_91 [ORI_BC_16] | Chloramphenicol |
| Plate 1 / G / 4 | pGL0_92 [ORI_BC_17] | Chloramphenicol |
| Plate 1 / G / 5 | pGL0_93 [ORI_BC_18] | Chloramphenicol |
| Plate 1 / G / 6 | pGL0_94 [ORI_BC_19] | Chloramphenicol |
| Plate 1 / G / 7 | pGL0_95 [ORI_BC_20] | Chloramphenicol |
| Plate 1 / G / 8 | pGL0_96 [J23100] | Chloramphenicol |
| Plate 1 / G / 9 | pGL0_97 [J23108] | Chloramphenicol |
| Plate 1 / G / 10 | pGL0_98 [J23114] | Chloramphenicol |
| Plate 1 / G / 11 | pGL0_99 [bsRBS] | Chloramphenicol |
| Plate 1 / G / 12 | pGL0_100 [Pbs] | Chloramphenicol |
| Plate 1 / H / 1 | pGL0_101 [PnptII] | Chloramphenicol |
| Plate 1 / H / 2 | pGL0_102 [pAgTEF1] | Chloramphenicol |
| Plate 1 / H / 3 | pGL0_103 [tAgTEF1] | Chloramphenicol |
| Plate 1 / H / 4 | pGL0_104 [pAnTrpC] | Chloramphenicol |
| Plate 1 / H / 5 | pGL0_105 [tAnTrpC] | Chloramphenicol |
| Plate 1 / H / 6 | pGL0_106 [TAmarelleT1] | Chloramphenicol |
| Plate 1 / H / 7 | pGL0_107 [TAmarelleT7] | Chloramphenicol |
| Plate 1 / H / 8 | pGL0_108 [TAmarelleT9] | Chloramphenicol |
| Plate 1 / H / 9 | pGL0_109 [TAmarelleT12] | Chloramphenicol |
| Plate 1 / H / 10 | pGL0_110 [pNG2] | Chloramphenicol |
| Plate 1 / H / 11 | pGL0_112 [oriT] | Chloramphenicol |
| Plate 1 / H / 12 | pGL0_113 [Dummy ORI] | Chloramphenicol |
| Plate 2 / A / 1 | pGL0_114 [p15A] | Chloramphenicol |
| Plate 2 / A / 2 | pGL0_115 [pBC1 ORI] | Chloramphenicol |
| Plate 2 / A / 3 | pGL0_121 [ORI_BC_21] | Chloramphenicol |
| Plate 2 / A / 4 | pGL0_122 [ORI_BC_22] | Chloramphenicol |
| Plate 2 / A / 5 | pGL0_123 [ORI_BC_23] | Chloramphenicol |
| Plate 2 / A / 6 | pGL0_124 [ORI_BC_24] | Chloramphenicol |
| Plate 2 / A / 7 | pGL0_125 [ORI_BC_25] | Chloramphenicol |
| Plate 2 / A / 8 | pGL0_126 [ORI_BC_26] | Chloramphenicol |
| Plate 2 / A / 9 | pGL0_127 [ORI_BC_27] | Chloramphenicol |
| Plate 2 / A / 10 | pGL0_128 [ORI_BC_28] | Chloramphenicol |
| Plate 2 / A / 11 | pGL0_129 [ORI_BC_29] | Chloramphenicol |
| Plate 2 / A / 12 | pGL0_130 [ORI_BC_30] | Chloramphenicol |
| Plate 2 / B / 1 | pGL0_142 [EilR-PjexD] | Chloramphenicol |
| Plate 2 / B / 2 | pGL0_143 [EilR-PjexH1] | Chloramphenicol |
| Plate 2 / B / 3 | pGL0_144 [EilR-PjexH2] | Chloramphenicol |
| Plate 2 / B / 4 | pGLEL_4 [BF] | Chloramphenicol |
| Plate 2 / B / 5 | pGLEL_5 [CF] | Chloramphenicol |
| Plate 2 / B / 6 | pGLDV_17 [380G] | Chloramphenicol |
| Plate 2 / B / 7 | pGLDV_13 [38e1G] | Chloramphenicol |
| Plate 2 / B / 8 | pGLDV_14 [38e2G] | Chloramphenicol |
| Plate 2 / B / 9 | pGLDV_15 [38e3G] | Chloramphenicol |
| Plate 2 / B / 10 | pGLDV_16 [38e4G] | Chloramphenicol |
| Plate 2 / B / 11 | pGLDV_20 [31e1G] | Chloramphenicol |
| Plate 2 / B / 12 | pGL2_141 | Chloramphenicol |
| Plate 2 / C / 1 | pGL2_144 | Chloramphenicol |
| Plate 2 / C / 2 | pGL1_7 | Kanamycin |
| Plate 2 / C / 3 | pGL1_8 | Kanamycin |
| Plate 2 / C / 4 | pGL1_9 | Kanamycin |
| Plate 2 / C / 5 | pGL1_35 | Kanamycin |
| Plate 2 / C / 6 | pGL1_36 | Kanamycin |
| Plate 2 / C / 7 | pGL1_37 | Kanamycin |
| Plate 2 / C / 8 | pGL1_38 | Kanamycin |
| Plate 2 / C / 9 | pGL1_39 | Kanamycin |
| Plate 2 / C / 10 | pGL1_40 | Kanamycin |
| Plate 2 / C / 11 | pGL1_41 | Kanamycin |
| Plate 2 / C / 12 | pGL1_42 | Kanamycin |
| Plate 2 / D / 1 | pGL1_43 | Kanamycin |
| Plate 2 / D / 2 | pGL1_44 | Kanamycin |
| Plate 2 / D / 3 | pGL1_45 | Kanamycin |
| Plate 2 / D / 4 | pGL1_46 | Kanamycin |
| Plate 2 / D / 5 | pGL1_47 | Kanamycin |
| Plate 2 / D / 6 | pGL1_48 | Kanamycin |
| Plate 2 / D / 7 | pGL1_59 | Kanamycin |
| Plate 2 / D / 8 | pGLEL_1 [βω] | Kanamycin |
| Plate 2 / D / 9 | pGLEL_2 [γω] | Kanamycin |
| Plate 2 / D / 10 | pGLEL_3 [εω] | Kanamycin |
| Plate 2 / D / 11 | pGLDV_21 [28o1*G] | Kanamycin |
| Plate 2 / D / 12 | pGLDO_1 [α-sfYFP-β] | Kanamycin |
| Plate 2 / E / 1 | pGL1_1 | Kanamycin |
| Plate 2 / E / 2 | pGL1_2 | Kanamycin |
| Plate 2 / E / 3 | pGL1_3 | Kanamycin |
| Plate 2 / E / 4 | pGLDV_5 [28o1G] | Kanamycin |
| Plate 2 / E / 5 | pGLDV_6 [28o2G] | Kanamycin |
| Plate 2 / E / 6 | pGLDV_7 [28o3G] | Kanamycin |
| Plate 2 / E / 7 | pGLDV_8 [28o4G] | Kanamycin |
| Plate 2 / E / 8 | pGLDV_42 [21o1G] | Kanamycin |
| Plate 2 / E / 9 | pGL1_4 | Kanamycin |
| Plate 2 / E / 10 | pGL1_5 | Kanamycin |
| Plate 2 / E / 11 | pGL1_6 | Kanamycin |
| Plate 2 / E / 12 | pGLDV_10 [18e2G] | Ampicillin |
| Plate 2 / F / 1 | pGLDV_11 [18e3G] | Ampicillin |
| Plate 2 / F / 2 | pGLDV_12 [18e4G] | Ampicillin |
| Plate 2 / F / 3 | pGLDV_19 [11e1G] | Ampicillin |
| Plate 2 / F / 4 | pGLDV_1 [18o1G] | Ampicillin |
| Plate 2 / F / 5 | pGLDV_2 [18o2G] | Ampicillin |
| Plate 2 / F / 6 | pGLDV_3 [18o3G] | Ampicillin |
| Plate 2 / F / 7 | pGLDV_4 [18o4G] | Ampicillin |
| Plate 2 / F / 8 | pGLDV_41 [11o1G] | Ampicillin |
| Plate 2 / F / 9 | pGLDV_9 [18e1G] | Ampicillin |
| Plate 2 / F / 10 | pGL2_136 | Chloramphenicol and Kanamycin |
| Plate 2 / F / 11 | pGL2_137 | Chloramphenicol and Tetracycline |
| Plate 2 / F / 12 | pGL2_142 | Chloramphenicol and Bleocin (Zeocin) |
| Plate 2 / G / 1 | pGL2_145 | Chloramphenicol and Erythromycin |
| Plate 2 / G / 2 | pGL0_17 [AMA1 short ORI] | Spectinomycin |
| Plate 2 / G / 3 | pGL2_135 | Chloramphenicol and Ampicillin |
| Plate 2 / G / 4 | pGL2_138 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 5 | pGL2_175 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 6 | pGL2_176 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 7 | pGL2_177 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 8 | pGL2_161 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 9 | pGL2_162 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 10 | pGL2_163 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 11 | pGL2_164 | Chloramphenicol and Gentamicin |
| Plate 2 / G / 12 | pGL2_165 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 1 | pGL2_166 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 2 | pGL2_167 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 3 | pGL2_168 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 4 | pGL2_169 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 5 | pGL2_170 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 6 | pGL2_171 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 7 | pGL2_172 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 8 | pGL2_173 | Chloramphenicol and Gentamicin |
| Plate 2 / H / 9 | pGL2_174 | Chloramphenicol and Gentamicin |
Kit Plate Map - #1000000234
Accessory Plasmids
Twenty-four additional Level 0, 1, and 2 plasmids are also available for use with this kit. These plasmids are not included in the kit, but can be ordered separately: