Bacterial Expression Systems
- Protein Purification
- Protein Visualization
- Protein Interactions
- Controlled Expression
- Reporter Plasmids
- Genome Engineering
Bacteria are commonly used to create, store, and replicate plasmids of all types, but beyond that, researchers also use bacteria as model systems to answer many interesting biological questions. Escherichia coli is the most widely used bacterial organism due to its ease of use, speed of growth, and well understood biology. Other bacterial models, like gram-positive bacteria, are also being used for their distinct features. Addgene distributes many plasmids that can help researchers plan their experiments using bacterial expression systems. Read below and browse our curated collection of bacterial expression plasmids to find out more!
Protein Purification
To study the function, structure, and activity of a protein it is generally necessary to purify it. Specific bacterial strains such as E. coli BL21(DE3) are used as biofactories to express plasmids and produce proteins in high yields. Proteins can then be extracted from the culture and purified through affinity chromatography. To facilitate the purification process, proteins are often expressed alongside epitope tags, which can also later be used for protein detection in western blots, or removed using protease cleavage sites. Other protein tags and signal peptides are used to enhance protein solubility and to direct recombinant proteins to the periplasmic space between the inner and outer membranes. Targeting proteins to the periplasm reduces bacterial protein contamination and facilitates extraction.
Browse our most popular plasmids for protein purification. Commonly used tags, cleavage sites, and signal peptides include:
- Epitope tags: 6xHis, Flag, Strep II, c-Myc, HA, V5, GST
- Solubility tags: MBP, SUMO, TrxA, Mocr, NusA
- Cleavage sites: TEV protease, factor Xa, enterokinase, thrombin
- Signal peptides for periplasmic localization: PelB, MalE, OmpA
| ID | Plasmid | Promoter | Tags | PI |
|---|
Additional Addgene Protein Purification Resources
- The Structural Genomics Consortium (SGC) collection page contains an extensive selection of plasmids and tags for protein purification.
- The pTD Plasmid Series contains plasmids suitable for replication in many gram-negative bacteria with different purification tag combinations, including PelB tags for localization to the periplasm.
- The pCri System (Addgene #1000000058) plasmids can be utilized for heterologous cytoplasmic and periplasmic protein expression in E. coli, Bacillus subtilis, and Pichia pastoris.
- The EcoFlex MoClo Toolkit (Addgene #1000000080) features a library of promoters and purification tags compatible with Golden Gate modular cloning (MoClo) for use in E. coli.
Addgene Blog
Protein Visualization
To determine the location of your protein of interest within the cell, you will need to tag it with something that makes it easy to see under the microscope. The plasmids in this collection are clone-in ready plasmids that contain fluorescent protein (FP) tags so you can express your protein of interest fused to FPs and monitor their localization in live cells.
| ID | Plasmid | Tags | PI |
|---|
Additional Addgene Protein Visualization Resources
- Visit our Fluorescent Protein Guide to find an extensive collection of FPs, including photoactivatable, photoconvertible, and photoswitchable proteins, organized by color and expression species.
- The New England Biolabs Cell-Imaging Plasmid Collection contains plasmids for non-toxic protein labeling and visualization inside living or fixed bacterial and mammalian cells.
Addgene Blog
Protein Interactions
Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) are techniques that have been widely used to study protein–protein interactions using fluorescence microscopy. FRET relies on the principle that when a donor fluorophore is excited, it can transfer its energy to an acceptor fluorophore if they are within a certain distance. You can test if two proteins bind or interact together by fusing each of them to two FPs whose emission and excitation spectra overlap. That way if your proteins of interest interact, they will bring both FPs together and you will be able to detect fluorescence both from the donor and the acceptor fluorophore by only exciting the donor. In BiFC, proteins of interest are fused to each part of a split FP. When the candidate proteins interact, the pieces of the FP can get together and reconstruct the full FP. Find plasmids containing FPs and split FPs that can be fused to your proteins of interest in bacterial FRET and BiFC studies.
| ID | Plasmid | Fluorescent Protein | Application | PI | |
|---|---|---|---|---|---|
|
14030 14031 |
CyPet (donor) YPet (acceptor) |
FRET | Patrick Daugherty | ||
|
18084 54856 |
mAmetrine1.1 (donor) tdTomato (acceptor) |
FRET/Dual FRET | |||
|
54553 54723 |
mTFP1 (donor) mCitrine (acceptor) |
FRET/Dual FRET | Robert Campbell, Michael Davidson | ||
|
54571 54856 |
mT-Sapphire (donor) tdTomato (acceptor) |
FRET | |||
|
54575 54771 |
Clover (donor) mRuby2 (acceptor) |
FRET | Michael Davidson | ||
| 18856 | pGWF1 |
ECFP (donor) Venus (acceptor) |
FRET | Wolf Frommer | |
| 65616 | pFLIP38 |
ECFP (donor) Citrine (acceptor) |
FRET | Wolf Frommer | |
| 65617 | pFLIP42 |
mCerulean (donor) Citrine (acceptor) |
FRET | Wolf Frommer | |
| 65618 | pFLIP43 |
ECFP (donor) mVenus (acceptor) |
FRET | Wolf Frommer | |
| 87856 | pET-BiFC | mVenus (reconstructed) | BiFC | Dan Mulvihill | |
| 39866 | pWA PAS-E BAD K-GAFm | iRFP (reconstructed) | BiFC | Vladislav Verkhusha | |
|
52732 52733 |
GFP (reconstructed) | BiFC | Lynne Regan | ||
|
168257 168472 |
DiB2 (reconstructed) | BiFC | Jens Meiler |
Additional Addgene Protein Interactions Resources
- Check out our Fluorescent Protein Guide for a selection of mammalian and bacterial FRET-related vectors and standards.
- The Hoschschild Bacterial Two-Hybrid (B2H) System allows you to fuse proteins of interest to transcriptional regulatory elements that will turn on gene expression upon protein–protein interaction.
- The cpFRET - FRET-based Biosensors Kit (Addgene #1000000021) is a vector library for the generation of FRET biosensors for ratiometric measurements.
- The MoBiFc Toolkit (Addgene #1000000222) is a collection of Golden Gate compatible vectors for bimolecular fluorescence complementation experiments in plants.
- The Zinc Finger Consortium Modular Assembly Kits (Addgene #1000000005, 1000000010) contain plasmids and an engineered strain for a B2H system used to screen the activity of arrays of zinc finger nucleases.
Addgene Blog
Controlled Expression
It isn’t always advantageous to constitutively express your gene of interest at high levels. Perhaps high expression leads to slow bacterial growth, or maybe you only want to study the effects of protein expression in the stationary phase. In these cases, you may want to control protein levels or to turn on expression only at a specific time. This collection contains plasmids whose expression levels can be controlled by a variety of small molecules, light, and temperature, in different bacterial systems.
| ID | Plasmid | Promoter | Inducer | Expression Species | PI | |
|---|---|---|---|---|---|---|
| 44249 | pdCas9-bacteria | pTetO | Anhydrotetracycline (aTc) | Escherichia coli | Stanley Qi | |
| 11518 | pDest-527 | T7-lacO | Lactose/IPTG | Escherichia coli | Dominic Esposito | |
| 43796 | pDawn | FixK2 | Blue light (470 nm) | Escherichia coli | Andreas Moeglich | |
| 68940 | pRMC2 | Pxyl/TetO | Anhydrotetracycline (aTc) | Staphylococcus aureus | Tim Foster | |
| 36267 | pBAD33.1 | pBAD | Arabinose | Escherichia coli | Christian Raetz | |
| 26098 | pCW-LIC | 3xPtac | Lactose/IPTG | Escherichia coli | Cheryl Arrowsmith | |
| 26094 | pET28a-LIC | T7-lacO | Lactose/IPTG | Escherichia coli | Cheryl Arrowsmith | |
| 43795 | pDusk | pR_FixK2 | Blue light (470 nm) | Escherichia coli | Andreas Moeglich | |
| 26092 | pET15-MHL | T7-lacO | Lactose/IPTG | Escherichia coli | Cheryl Arrowsmith | |
| 46886 | pMSP3535 | PnisA | Nisin | Escherichia coli, gram-positive bacteria | Gary Dunny | |
| 74064 | pZ8-Ptac | Ptac + LacI | Lactose/IPTG | Corinebacterium glutamicum | Timothy Lu | |
| 17972 | pSE100 | Pmyc1/TetO | Anhydrotetracycline (aTc) | Escherichia coli, Mycobacterium tuberculosis | Sabine Ehrt | |
| 44561 | pST-KT | Pmyc1/TetO | Anhydrotetracycline (aTc) | Escherichia coli, Mycobacterium tuberculosis | Vinay Nandicoori | |
| 78577 | pL99 | PnitA-NitR | ε-caprolactam | Escherichia coli, Streptomyces sp. | Xuming Mao | |
| 46888 | pMSP3545 | PnisA | Nisin | Escherichia coli, gram-positive bacteria | Gary Dunny | |
| 48095 | pQF | PQ5 | p-isopropyl benzoate (cumate) | Sphingomonas sp., Caulobacter crescentus, Paracoccus denitrificans, Methylobacterium extorquens | Julia Vorholt | |
| 84693 | pMyNT-kan | Acetamidase promoter | Acetamide | Escherichia coli, Mycobacterium smegmatis | Matthias Wilmanns | |
| 84692 | pMyC-kan | Acetamidase promoter | Acetamide | Escherichia coli, Mycobacterium smegmatis | Matthias Wilmanns | |
| 84689 | pMyBADC-kan | pBAD | Arabinose | Escherichia coli, Mycobacterium smegmatis | Matthias Wilmanns | |
| 17806 | pPro18 | pPrpB | Propionate | Escherichia coli | Jay Keasling | |
| 17810 | pPro33 | pPrpB | Propionate | Escherichia coli | Jay Keasling | |
| 113634 | pSCrhaB2 | PrhaB | Rhamnose | Burkholderia cenocepacia | Miguel Valvano | |
| 47640 | pCS-PesaRlux | PesaR | 3OC6HSL (Quorum sensing molecule) | Escherichia coli | Cynthia Collins | |
| 84689 | pMyBADC-kan | pBAD | Arabinose | Escherichia coli, Mycobacterium smegmatis | Matthias Wilmanns | |
| 44447 | pLC290 | pR/cmtO | p-isopropyl benzoate (cumate) | Methylobacterium extorquens | Christopher Marx | |
| 44448 | pLC291 | pR/TetO | Anhydrotetracycline (aTc) | Methylobacterium extorquens | Christopher Marx | |
| 122635 | pPEPZ-Plac | Plac | Lactose/IPTG | Streptococcus pneumoniae | Jan-Willem Veening | |
| 188978 | pREDawn-AmpR-MCS | FixK2 | Red light (660 nm) | Escherichia coli | Heikki Takala | |
| 31395 | pJT118 | PcpcG2 | Green Light (532 nm) | Escherichia coli | Christopher Voigt | |
| 164226 | pSCrhaB2plus | PrhaBAD | Rhamnose | Burkholderia sp. | Silvia Cardona | |
| 44565 | pST-KNarK2 | Pnark2 | Hypoxia | Escherichia coli, Mycobacterium tuberculosis | Vinay Nandicoori | |
| 188974 | pREDusk-AmpR-MCS | FixK2 | Red light (660 nm) | Escherichia coli | Heikki Takala | |
| 47641 | pCS-PluxIlux | PluxI | LuxR | Escherichia coli | Cynthia Collins | |
| 222348 | pJMP3653 | PabstBR | IPTG | Escherichia coli, Acinetobacter baumannii | Jason Peters | |
| 127088 | pMS17 | tcp830 | Anhydrotetracycline (aTc) | Streptomyces sp. | Maggie Smith | |
| 74065 | pZ8-Prp | PprpD2 | Propionate | Corinebacterium glutamicum | Timothy Lu | |
| 36252 | pRibo EcoHind | Riboswitch | Teophylline | Escherichia coli, Mycobacterium smegmatis | Carolyn Bertozzi, Jessica Seeliger | |
| 36251 | pRibo BsaHind | Riboswitch | Teophylline | Escherichia coli, Mycobacterium smegmatis | Carolyn Bertozzi, Jessica Seeliger | |
| 202406 | pJYP1 | Riboswitch | Cobalamin (vitamin B12) | Escherichia coli | Remy Loris | |
| 47663 | pAC-EsaR-I70V | Plac | 3OC6HSL (Quorum sensing molecule) | Escherichia coli | Cynthia Collins | |
| 112197 | pANY3 | Temperature (42 °C) | Escherichia coli | Yingfeng An |
Other Addgene Controlled Expression Resources
- Check out our Tetracycline (Tet) Inducible Expression Collection for an extensive selection of plasmids using tetracycline-regulated transcriptional activation or repression.
- The BIOFAB plasmid set (Addgene #1000000037) contains specific bacterial promoters that vary in strength and that can be used to reliably drive expression of a gene of interest in E. coli.
- The Marionette Sensor Collection (Addgene #1000000137) allows you to independently control the expression of up to twelve genes using small-molecule inducers in the same E. coli strain.
Addgene Blog
Reporter Plasmids
Reporter plasmids can be used to detect events and molecules in and around a bacterium. Plasmids containing easily measurable reporter genes (e.g., LacZ, luciferase, or fluorescent proteins) under the control of a promoter element can be used to quickly determine whether or not a particular protein can activate expression from that element. When this activation requires a transcription factor to bind to a small molecule, the reporter plasmid can be used to detect the presence of that small molecule and sometimes even quantify it. Bacterial one-hybrid (B1H) systems also use reporter genes to identify interactions between proteins and specific DNA sequences.
| ID | Plasmid | Reports | Reporter Mechanism | Expression Species | PI | |
|---|---|---|---|---|---|---|
| 14474 | pRU1161 | Promoter activity | GUS activity and fluorescence (mRFP1) | Gram-negative bacteria | Philip Poole | |
| 14473 | pRU1156 | Promoter activity | GUS activity and fluorescence (GFPmut3.1) | Gram-negative bacteria | Philip Poole | |
| 14471 | pRU1144 | Promoter activity | Fluorescence (mRFP1) | Gram-negative bacteria | Philip Poole | |
| 14462 | pRU1097 | Promoter activity | Fluorescence (GFPmut3.1) | Gram-negative bacteria | Philip Poole | |
| 14461 | pRU1701 | Promoter activity | Fluorescence (GFP+) | Gram-negative bacteria | Philip Poole | |
| 14460 | pOT2 | Promoter activity | Fluorescence (GFPuv) | Gram-negative bacteria | Philip Poole | |
| 14083 | pAKgfplux1 | Promoter activity | Fluorescence (GFPmut3a) and luminescence (lux operon) | Gram-negative bacteria | Attila Karsi | |
| 14080 | pAKlux2 | Promoter activity | Luminescence (lux operon) | Gram-negative bacteria | Attila Karsi | |
| 14076 | pAKgfp1 | Promoter activity | Fluorescence (GFPmut3a) | Gram-negative bacteria | Attila Karsi | |
| 18855 | FLIParaF.Ec-250n | Arabinose | FRET fluorescence (CFP and Venus) | Escherichia coli | Wolf Frommer | |
| 20336 | pRsetB-his7-Perceval | ATP:ADP ratio | Fluorescence (GFP) | Escherichia coli | Gary Yellen | |
| 187836 | pVoPo-01 | Promoter activity | Fluorescence (mCherry) | Fusobacterium nucleatum | Jörg Vogel | |
| 46002 | pGR | Terminator strength | Fluorescence (GFP:mRFP1 ratio) | Escherichia coli | Christopher Voigt | |
| 65008 | pCRISPReporter-mCherry | Promoter activity | Fluorescence (mCherry) | Escherichia coli | Mattheos Koffas | |
| 173481 | HC-M | ATP | Fluorescence (GFPmut2) | Escherichia coli | Rahul Sarpeshkar | |
| 111614 | pCdrA-gfpC | Cyclic di-GMP | Fluorescence (GFPmut2) | Pseudomonas aeruginosa | Tim Tolker-Nielsen | |
| 90217 | pMV762-Peredox-mCherry | NADH:NAD+ ratio | Fluorescence (mCherry) | Mycobacterium sp. | Ashwani Kumar | |
| 134405 | pBbAW4k-loxP-TT-loxP-mRFP1 | Cre recombinase activity | Fluorescence (mRFP1) | Escherichia coli | Mary Dunlop | |
| 124605 | pSCM001 | Cytoplasmic pH | Fluorescence (mCherry) | Escherichia coli | David Summers | |
| 24659 | pCHERRY3 | Promoter activity | Fluorescence (mCherry) | Mycobacterium sp. | Tanya Parish | |
| 24657 | pASTA3 | Promoter activity | Fluorescence (tdTomato) | Mycobacterium sp. | Tanya Parish | |
| 24658 | pCHARGE3 | Promoter activity | Fluorescence (turbo-365) | Mycobacterium sp. | Tanya Parish | |
| 26156 | pMV306hsp+FFluc | Promoter activity | Luminescence (firefly luciferase) | Mycobacterium sp. | Brian Robertson, Siouxsie Wiles | |
| 26161 | pMV306hsp+LuxG13 | Promoter activity | Luminescence (lux operon) | Mycobacterium sp. | Brian Robertson, Siouxsie Wiles | |
| 26159 | pMV306hsp+Lux | Promoter activity | Luminescence (lux operon) | Mycobacterium sp. | Brian Robertson, Siouxsie Wiles | |
| 26160 | pMV306G13+Lux | Promoter activity | Luminescence (lux operon) | Mycobacterium sp. | Brian Robertson, Siouxsie Wiles | |
| 106476 | pET28a_T5-ARG1 | Bacterial cells | Ultrasound (acoustic reporter gene, ARG1) | Escherichia coli Nissle 1917 | Mikhail Shapiro | |
| 106473 | pET28a_T7-ARG1 | Bacterial cells | Ultrasound (acoustic reporter gene, ARG1) | Escherichia coli | Mikhail Shapiro | |
| 106474 | pET28a_T7-ARG2 | Bacterial cells | Ultrasound (acoustic reporter gene, ARG2) | Escherichia coli | Mikhail Shapiro | |
| 192473 | pBAD-bARGSer-AxeTxe | Bacterial cells | Ultrasound (acoustic reporter gene, Serratia ARG) | Escherichia coli | Mikhail Shapiro | |
| 78565 | pCM18 | Promoter activity | Luminescence (lux operon) | Vibrio cholerae | James Kaper |
Additional Addgene Reporter Plasmids Resources
- The Wolfe Bacterial One-Hybrid (B1H) System can be used for determining the DNA-binding specificity of proteins such as transcription factors.
Addgene Blog
Genome Engineering
Modifying the bacterial genome by knocking out genes or introducing specific mutations can reveal important insights into gene function. These genetic changes also allow for finer control over protein expression and activity, helping to conserve cellular resources otherwise spent on producing high amounts of protein. Whether you're investigating basic bacterial gene biology or engineering metabolic pathways to synthesize a target metabolite, genetic tools like CRISPR-Cas9 are not only valuable but often essential.
Find a collection of CRISPR plasmids that have been designed for their use in bacteria in our Bacterial CRISPR Plasmids page.
Additional Addgene Genome Engineering Resources
- Learn more about CRISPR with Addgene's CRISPR Guide.
- Browse CRISPR plasmids available at Addgene through our CRISPR Plasmids and Resources page.
- Check our Cre-Lox Plasmids Collection for a selection of plasmids containing recombinases to induce targeted genetic inversion, deletions, and translocations events.
- Visit our TALEN Collection page to find plasmids and kits for efficient assembly of TALEN constructs.
- The Zinc Finger Consortium page contains a variety of reagents for engineering and expressing zinc finger proteins.
Addgene Blog
Content last reviewed on 17 June 2025.
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