Engineering BioBrick vectors from BioBrick parts

Abstract

Background: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts.

Results: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts.

Conclusion: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.

Figure 1

The BioBrick base vector (BBa_I51020). Schematic diagram of BBa_I51020: a BioBrick base vector designed to facilitate construction of new BioBrick vectors. Parts from the collection listed in Figure 5 were used to construct BBa_I51020.

Figure 2

How to build new BioBrick vectors. Assembly strategy for a new BioBrick vector using the BioBrick base vector BBa_I51020. (A) The replication origin and antibiotic resistance cassette should each be BioBrick standard biological parts. (B) Assemble the desired replication origin and antibiotic resistance cassette via BioBrick standard assembly to construct a composite origin and antibiotic resistance cassette. (C) Digest the resulting BioBrick composite part with XbaI and SpeI. (D) To excise the ampicillin resistance marker, digest the base vector with NheI. XbaI, SpeI, and NheI all generate compatible cohesive DNA ends that, when ligated with a DNA end from a one of the other enzymes, produce a non-palindromic sequence that cannot be cut by any of the three enzymes. Finally, ligate the digested composite origin and resistance marker to the digested base vector. (E) The result is the new BioBrick vector pSB4K5-I52002.

Figure 3

How to use a new BioBrick vector for standard assembly. Assembly strategy for two BioBrick standard biological parts using a new BioBrick vector. (A) Digest the prefix part with enzymes EcoRI and SpeI. (B) Digest the suffix part with restriction enzymes XbaI and PstI. (C) Digest the destination vector (pSB4K5-I52002) into which the two parts will be assembled with restriction enzymes EcoRI and PstI. Without agarose gel purification of the linearized DNA, ligate the three fragments, transform into E. coli and plate on LB agar plates supplemented with the antibiotic corresponding to the destination vector resistance marker. (D) Most of the resulting colonies contain the composite BioBrick part cloned into the destination vector.

Figure 4

Using the new BioBrick vectors. To verify the function of the new BioBrick vectors, we performed a colony PCR using primers that anneal to the verification primer binding sites. To check the length of the resulting PCR products, we electrophoresed the reactions through an 0.8% agarose gel. Lanes 1–8 are the PCR products resulting from the amplification of the following BioBrick parts cloned into new BioBrick vectors. The desired PCR product lengths are in parentheses. Lane 1 is pSB4A5-I52001 (1370 bp), lane 2 is pSB4K5-T9003 (1883 bp), lane 3 is pSB4C5-E0435 (814 bp), lane 4 is pSB4T5-P20061 (2988 bp), lane 5 is pSB3K5-I52002 (1370 bp), lane 6 is pSB3C5-I52001 (1370 bp), lane 7 is pSB3T5-I6413 (867 bp), and lane 8 is BBa_I51020 (1370 bp). Lane 9 is 1 μg of 2-log DNA ladder (New England Biolabs, Inc.). The 0.5 kb, 1 kb, and 3 kb DNA fragments in the DNA ladder are annotated.

Figure 5

New BioBrick vector parts. The Registry part number, function, and graphical notation of each constructed BioBrick vector part are listed. The part collection includes (1) BBa_G00000: BioBrick cloning site prefix including the EcoRI (E) and XbaI (X) restriction enzyme sites, (2) BBa_G00001: BioBrick cloning site suffix including the SpeI (S) and PstI (P) restriction enzyme sites which, together with the BioBrick prefix, forms a BioBrick cloning site for compatibility with all BioBrick standard biological parts, (3) BBa_P1016: positive selection marker ccdB to improve yield of insert-containing clones during part assemblies, (4) BBa_I50022: pUC19-derived high copy replication origin within the BioBrick cloning site that allows for easy plasmid DNA purification of the base vector and any derived vectors, (5) BBa_B0042: a short DNA sequence that has translational stop codons in all six reading frames to prevent translation into or out of the BioBrick cloning site, (6) BBa_B0053-B0055 and BBa_B0062: forward and reverse transcriptional terminators flanking the BioBrick cloning site to prevent transcription into or out of the BioBrick cloning site, (7) BBa_G00100 and BBa_G00102: sequence verification primer annealing sites for primers VF2 and VR, (8) BBa_B0045: NheI (N) restriction site for insertion of desired replication origin and resistance marker to construct vector of interest, (9) BBa_P1006: ampicillin resistance selection marker to facilitate propagation of the base vector, (10) BBa_P1002-P1005: four antibiotic resistance markers, and (11) BBa_I50042 and BBa_I50032: pSC101 and p15A replication origins. Each part is used either as a component of the BioBrick base vector BBa_I51020 (1–9) or to construct new BioBrick vectors (10–11).