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EcoFlex MoClo Kit
(Kit # 1000000080 )

Depositing Lab:   Paul Freemont

The EcoFlex Toolkit is a collection of plasmids that features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins for use in E. coli for a variety of applications.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Add to Cart
$350 USD + shipping

Available to academics and nonprofits only.

Original Publication

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. Moore SJ, Lai HE, Kelwick R, Chee SM, Bell D, Polizzi KM, Freemont PS. ACS Synth Biol. 2016 May 2. [Epub ahead of print] PubMed PMID: 27096716 .

Description

EcoFlex is a Golden Gate kit for E. coli synthetic biology. The kit consists of a modular library that uses the MoClo assembly standard for use in a variety of applications, like recombinant protein purification and cell-free circuit design.

Figure 1A from Moore et al
Figure 1B from Moore et al

EcoFlex assembly. (A) Level 0 parts are held in pBP (bioparts–promoters, RBS, tags and terminators) and pBP-ORF (open reading frames) plasmids and assembled with BsaI and T4 DNA ligase into pTU1 destination plasmids (A, -B, -C, -D or -D1, -E) forming Level 1 TUs. Genes are cloned with NdeI (5′) and BamHI (3′), with the NdeI site providing an overlap between the start codon and RBS fusion site (CATA). Alternatively, if these sites are located within the gene, custom designed BsaI primers are used.

Level 2 modules (2–5 TUs) were assembled with BsmBI and T4 DNA ligase into pTU2 plasmids, which were modified to provide flexibility for assembling 3–5 TUs into a module. In addition, Level 3 pathways (up to 20 TUs) can be assembled from compatible Level 2 modules into pTU3-A or pTU3-B using BsaI and T4 DNA ligase.

(B) Assembly options (i) for purification tags and promoter choice, (ii) fusion site sequences and positions for Level 1 assembly of TUs.

(C) Secondary module cloning for optimizing library screening and minimizing pathway variation. Table details compatible fusion sites and assembly options.

Figure 1 from Moore et al, 2016. PubMed PMID: 27096716 . Courtesy of ACS Synthetic Biology.

EcoFlex kit graphical abstract

Graphical abstract from Moore et al, 2016. PubMed PMID: 27096716 . Courtesy of ACS Synthetic Biology.

Depositor-provided Files

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 EcoFlex kit was a gift from Paul Freemont (Addgene kit #1000000080).”

For your Reference section:

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. Moore SJ, Lai HE, Kelwick R, Chee SM, Bell D, Polizzi KM, Freemont PS. ACS Synth Biol. 2016 May 2. [Epub ahead of print] PubMed PMID: 27096716 .

EcoFlex MoClo Kit - #1000000080

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. 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

Ampicillin
Chloramphenicol

Inventory

Well Plasmid Resistance
A / 1 pTU1
Ampicillin
A / 2 pTU1-A-lacZ
Ampicillin
A / 3 pTU1-B-lacZ
Ampicillin
A / 4 pTU1-C-lacZ
Ampicillin
A / 5 pTU1-D-lacZ
Ampicillin
A / 6 pTU1-A-RFP
Ampicillin
A / 7 pTU1-B-RFP
Ampicillin
A / 8 pTU1-C-RFP
Ampicillin
A / 9 pTU1-D-RFP
Ampicillin
A / 10 pTU1-D1-RFP
Ampicillin
A / 11 pTU1-E-RFP
Ampicillin
A / 12 pTU3-A-RFP
Ampicillin
B / 1 pTU3-B-RFP
Ampicillin
B / 2 pBP
Chloramphenicol
B / 3 pBP-lacZ
Chloramphenicol
B / 4 pBP-ORF
Chloramphenicol
B / 5 pTU2-A-lacZ
Chloramphenicol
B / 6 pTU2-B-lacZ
Chloramphenicol
B / 7 pTU2-C-lacZ
Chloramphenicol
B / 8 pTU2-D-lacZ
Chloramphenicol
B / 9 pTU2-A-RFP
Chloramphenicol
B / 10 pTU2-B-RFP
Chloramphenicol
B / 11 pTU2-C-RFP
Chloramphenicol
B / 12 pTU2-D-RFP
Chloramphenicol
C / 1 pTU2-a-RFP
Chloramphenicol
C / 2 pTU2-b-RFP
Chloramphenicol
C / 3 pBP-ORF-eGFP
Chloramphenicol
C / 4 pBP-ORF-eCFP
Chloramphenicol
C / 5 pBP-ORF-mCherry
Chloramphenicol
C / 6 pBP-J23100
Chloramphenicol
C / 7 pBP-J23108
Chloramphenicol
C / 8 pBP-J23114
Chloramphenicol
C / 9 pBP-SJM901
Chloramphenicol
C / 10 pBP-SJM902
Chloramphenicol
C / 11 pBP-SJM903
Chloramphenicol
C / 12 pBP-SJM905
Chloramphenicol
D / 1 pBP-SJM906
Chloramphenicol
D / 2 pBP-SJM908
Chloramphenicol
D / 3 pBP-SJM910
Chloramphenicol
D / 4 pBP-SJM911
Chloramphenicol
D / 5 pBP-SJM912
Chloramphenicol
D / 6 pBP-SJM914
Chloramphenicol
D / 7 pBP-SJM915
Chloramphenicol
D / 8 pBP-T7
Chloramphenicol
D / 9 pBP-T7_RBS
Chloramphenicol
D / 10 pBP-T7_RBS-His6-Thrombin
Chloramphenicol
D / 11 pBP_BBa_B0034
Chloramphenicol
D / 12 pBP-pET-RBS
Chloramphenicol
E / 1 pBP-Tag_linker
Chloramphenicol
E / 2 pBP-HexHis
Chloramphenicol
E / 3 pBP-Strep(II)
Chloramphenicol
E / 4 pBP-TL1
Chloramphenicol
E / 5 pBP-TL2
Chloramphenicol
E / 6 pBP-TL3
Chloramphenicol
E / 7 pBP-TL4
Chloramphenicol
E / 8 pBP-TL5
Chloramphenicol
E / 9 pBP-TL6
Chloramphenicol
E / 10 pBP-TL7
Chloramphenicol
E / 11 pBP-TL8
Chloramphenicol
E / 12 pBP-TL9
Chloramphenicol
F / 1 pBP-TL10
Chloramphenicol
F / 2 pBP-TL11
Chloramphenicol
F / 3 pBP-TL12
Chloramphenicol
F / 4 pBP-BBa_B0012
Chloramphenicol
F / 5 pBP-BBa_B0015
Chloramphenicol
F / 6 pBP-L3S2P21
Chloramphenicol
F / 7 pBP-L3S1P51
Chloramphenicol
F / 8 pBP-L3S1P32
Chloramphenicol
F / 9 pBP-L3S1P11
Chloramphenicol
F / 10 pBP-L2U8H11
Chloramphenicol
F / 11 pBP-L2U5H11
Chloramphenicol
F / 12 pBP-L2U5H08
Chloramphenicol
G / 1 pBP-L2U3H03
Chloramphenicol
G / 2 pBP-L2U2H09
Chloramphenicol
G / 3 pBP-L1U1H09
Chloramphenicol
G / 4 pBP-L1U1H08
Chloramphenicol
G / 5 pTU2_2a
Chloramphenicol
G / 6 pTU2_2b
Chloramphenicol
Data calculated @ 2017-09-24

Kit Plate Map - #1000000080

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