Constructing new BioBrick vectors using the BioBrick base vector

Constructing new BioBrick vectors starting from the BioBrick base vector requires just two assembly steps (Figure ​(Figure2).2). The replication origin and antibiotic resistance marker should each be BioBrick standard parts. To construct a BioBrick vector, assemble the origin and antibiotic resistance marker via BioBrick standard assembly (first assembly step). Then, digest the resulting composite part with restriction enzymes XbaI and SpeI, and digest the BioBrick base vector with NheI to excise the ampicillin resistance marker. Next, ligate the composite origin and resistance marker to the linearized base vector (second assembly step). XbaI, SpeI, and NheI all generate compatible DNA ends that, when ligated with a DNA end from one of the other enzymes, produce a non-palindromic sequence that cannot be cut by any of the three enzymes. Thus, proper assembly of the vector eliminates any BioBrick enzyme sites and ensures that the resulting vector adheres to the BioBrick physical composition standard. Finally, transform the ligation product into a strain tolerant of ccdB expression, such as E. coli strain DB3.1 [48,49].

Open in a separate window

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.

To support the construction of new BioBrick vectors, we built four new antibiotic resistance markers and two replication origins all as BioBrick standard biological parts. The four antibiotic resistance markers express proteins that confer resistance to ampicillin (BBa_P1002 [Genbank:EU496092]), kanamycin (BBa_P1003 [Genbank:EU496093]), chloramphenicol (BBa_P1004 [Genbank:EU496094]), and tetracycline (BBa_P1005 [Genbank:EU496095]), respectively [50-53]. The two replication origins were derived from the pSC101 (BBa_I50042 [Genbank:EU496096]) and p15A (BBa_I50032 [Genbank:EU496097]) replicons, respectively [54,55]. We used the described procedure, base vector, and new vector parts to construct seven new BioBrick vectors: pSB4A5-I52002, pSB4K5-I52002, pSB4C5-I52002, pSB4T5-I52001, pSB3K5-I52002, pSB3C5-I52001, and pSB3T5-I52001 [Genbank:EU496098–EU496104].

Design of new BioBrick vectors parts

To adhere to the BioBrick standard for physical composition, BioBrick vector parts need only be free of the BioBrick restriction enzyme sites. However, we chose to design anew all BioBrick vector parts (Figure ​(Figure5),5), so that we could completely specify their DNA sequences. We compiled a list of potentially useful endonuclease sites for removal from all new BioBrick vector parts (Table ​(Table1).1). We targeted each group of endonuclease sites for removal for a different reason. We targeted recognition sites of enzymes that produce compatible cohesive ends to the BioBrick enzymes because such enzymes often prove useful in constructing new variants of BioBrick vectors. We targeted offset cutter sites because they may be useful in alternative restriction enzyme-based assembly methods [57]. We targeted homing endonuclease sites because they are commonly used in genome engineering [58]. We targeted some nicking endonuclease sites because they can be useful for specialized cloning applications [59]. Finally, we targeted several additional restriction endonuclease sites to keep them available for use by new standards for physical composition. Our list of endonuclease sites constitutes a set of target sequences that should be considered for removal from any newly synthesized BioBrick part, if possible. The target sequence set will change as the synthetic biology community develops new standards for physical composition of BioBrick parts. Some of the targeted endonuclease sites were naturally absent from the DNA sequences encoding our new vector parts. For any remaining sites, we removed the recognition sequences from the BioBrick vector parts by introducing point mutations. However, the functions of the pSC101 and pUC19-derived plasmid replication origins were sensitive to introduced mutations, so the replication origins used in this work are not free of all targeted endonuclease sites (see Methods). Similarly, issues during synthesis led to an unnecessary redesign of the ccdB positive selection marker, so it too is not free of all targeted endonuclease sites.

Open in a separate window

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).

Construction of BioBrick vector parts

We contracted for DNA synthesis of the four antibiotic resistance markers and the pSC101 replication origin to the DNA synthesis company Codon Devices, Inc. in Cambridge, MA. The four antibiotic resistance markers (BBa_P1002-P1005) were easily synthesized as designed. Testing confirmed that the four markers conferred resistance to the corresponding antibiotics. Synthesis of the pSC101 origin was also straightforward. However, testing revealed that our design for the pSC101 origin (BBa_I50040) was nonfunctional as a replication origin. We successfully reconstructed a functional pSC101 replication origin (BBa_I50042) via PCR of an existing plasmid. Thus, we presume that one or more of the introduced point mutations to eliminate endonuclease sites were deleterious to the plasmid replication function of the designed origin. We did not attempt to synthesize the p15A replication origin (BBa_I50032). Instead, like the pSC101 origin, we constructed p15A origin by PCR of an existing plasmid.

We constructed the functional pSC101 replication origin by PCR using pSB4A3-P1010 as a template and amplification primers I50042-f (5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG CTG TCA GAC CAA GTT TAC GAG-3') and I50042-r (5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAG TTA CAT TGT CGA TCT GTT C-3'). We constructed the p15A replication origin by PCR using pSB3K3-P1010 as a template and amplification primers I50032-f (5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATG GAA TAG ACT GGA TGG AG-3') and I50032-r (5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAA ACA CCC CTT GTA TTA CTG-3'). Each reaction was a mix of 45 μL PCR SuperMix High Fidelity, 31.25 pmoles each of forward and reverse primer, and 1 ng template DNA in a 50 μL total volume. The PCR conditions were an initial denaturation step of 95°C for 15 mins followed by 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 2.5 minutes. Finally, the reactions were incubated at 68°C for 20 minutes. We then added 20 units DpnI restriction enzyme to each reaction to digest the template DNA. The reactions were incubated for 2 hours at 37°C and then heat-inactivated for 20 minutes at 80°C. We purified both reactions using a MinElute PCR Purification kit according to the manufacturer's directions (QIAGEN, Germany). The pSC101 and p15A origin PCR products were used directly for assembly of the BioBrick vectors.

Construction of BioBrick base vector

We also contracted for synthesis of the entire BioBrick base vector. However, we encountered two issues during synthesis of the base vector. First, troubleshooting efforts during synthesis compromised the design of the base vector: failed attempts to clone the base vector into an E. coli strain intolerant of expression of the toxic protein CcdB led to an unnecessary redesign of the ccdB positive selection marker in the BioBrick base vector (from BBa_P1011 to BBa_P1016 [Genbank:EU496090]). Second, faulty part design adversely impacted the synthesis process: our pUC19-based replication origin design was similarly nonfunctional, so the base vector could not be propagated as specified. Yet, synthesized DNA for the BioBrick base vector was nevertheless provided. We eventually determined that the provided DNA was actually a fusion of two slightly different copies of the base vector: one with the designed, nonfunctional version of the pUC19 origin (BBa_I50020) and one with a functional version of the pUC19 origin (BBa_I50022 [Genbank:EU496091]). To obtain a single, corrected version of the BioBrick base vector, we performed a restriction digest of the provided base vector DNA with EcoRI. We then re-ligated 1 μL of a ten-fold dilution of the linearized base vector DNA. For detailed reaction conditions, see Assembly of BioBrick parts using the new BioBrick vectors. We transformed the religated BioBrick base vector into E. coli strain DB3.1 via electroporation and plated the transformed cells on LB agar plates supplemented with 100 μg/mL ampicillin to obtain the corrected BioBrick base vector BBa_I51020 [48,61,62]. Correct construction of the BioBrick base vector was verified by DNA sequencing by the MIT Biopolymers Laboratory.

Assembly of BioBrick vectors

We assembled the new BioBrick vectors as described (Figure ​(Figure2).2). For detailed reaction conditions, see Assembly of BioBrick parts using the new BioBrick vectors. However, we used the synthesized BioBrick base vector BBa_I51019 instead of the corrected BioBrick base vector BBa_I51020, since, at the time, we had not yet identified the issue with the provided synthesized DNA. As a result, we obtained a mixture of new vectors. Four of the constructed vectors have a functional version of the pUC19 origin (BBa_I50022) in the BioBrick cloning site and propagate at high copy (vectors with BBa_I52002: pSB4A5, pSB4K5, pSB4C5, and pSB3K5). The other three vectors have a nonfunctional version of the pUC19 origin (BBa_I50020) in the BioBrick cloning site and propagate at low copy (vectors with BBa_I52001: pSB4T5, pSB3C5, and pSB3T5). We chose to describe all seven vectors here for two reasons. First, all seven new BioBrick vectors can be used for the propagation and assembly of BioBrick parts; the vectors pSB4T5, pSB3C5, and pSB3T5 are just slightly less convenient for plasmid DNA purification. Second, the difficulties that we encountered during construction of the BioBrick base vector are illustrative of the current interdependence of DNA design and DNA synthesis (see Discussion).

Assembly of BioBrick parts using the new BioBrick vectors

We assembled BioBrick composite parts as described (Figure ​(Figure3).3). We performed all restriction digests by mixing 0.5–1 μg DNA, 1X NEBuffer 2, 100 μg/ml Bovine Serum Albumin, and 1 μL each needed restriction enzyme in a 50 μL total volume. Restriction digest reactions were incubated for at least 2 hours at 37°C and then heat-inactivated for 20 minutes at 80°C. We then dephosphorylated the destination vector into which the parts were assembled. (When assembling BioBrick vectors, we dephosphorylated the composite origin and resistance marker to prevent circularization of this DNA fragment.) We performed dephosphorylation reactions by adding 5 units Antarctic Phosphatase and 1X Antarctic Phosphatase Reaction Buffer in a total volume of 60 μL to the heat-inactivated restriction digest reaction. We incubated dephosphorylation reactions for 1 hour at 37°C and inactivated the phosphatase by heating to 65°C for 5 minutes. We purified all reactions using a MinElute PCR Purification kit according to the manufacturer's directions (QIAGEN). We performed all ligation steps by mixing 2–4 μL of each purified, linearized DNA, 1X T4 DNA Ligase Reaction Buffer, and 200 units T4 DNA Ligase in a 10 μL total volume. We incubated the ligation reactions for 20 minutes at room temperature. We transformed all assembled BioBrick parts into E. coli strain TOP10 via chemical transformation [63-65]. (We transformed the assembled BioBrick vectors into E. coli strain DB3.1 via electroporation [48,61,62].) Transformed cells were plated on LB agar plates supplemented with 100 μg/mL ampicillin, 50 μg/mL kanamycin, 35 μg/mL chloramphenicol, or 15 μg/mL tetracycline as appropriate. We identified clones with correct construction of BioBrick parts by growth on the plates supplemented with the correct antibiotic, lack of growth on plates supplemented with other antibiotics, length verification by colony PCR (see next section), and DNA sequencing by the MIT Biopolymers Laboratory.

Verification of correct BioBrick part assembly via colony PCR

To demonstrate the correct assembly of BioBrick parts using the new BioBrick vectors, we performed a colony PCR using primers that anneal to the verification primer binding sites. We picked one colony and diluted it into 100 μL water. Then we mixed 9 μL PCR SuperMix High Fidelity, 6.25 pmoles VF2 primer (5'-TGC CAC CTG ACG TCT AAG AA-3'), 6.25 pmoles VR primer (5'-ATT ACC GCC TTT GAG TGA GC-3'), and 1 μL colony suspension. The PCR conditions were as described previously but using an annealing temperature of 62°C and an elongation time of 3.5 minutes. We diluted the reactions four-fold with water and then performed an agarose gel electrophoresis of 20 μL of each diluted reaction using a 0.8% E-Gel^®. We also electrophoresed 1 μg of 2-log DNA ladder (New England Biolabs, Inc., Ipswich, MA) to verify the length of each PCR product. The gel was imaged with 302 nm transilluminating ultraviolet light using an ethidium bromide emission filter and an exposure time of 614 milliseconds.

Materials for all PCR and agarose gel electrophoresis steps in this work were purchased from the Invitrogen Corporation in Carlsbad, CA unless otherwise specified. Reagents for all restriction digest, dephophorylation, and ligation reactions were purchased from New England Biolabs, Inc., Ipswich, MA. All PCR and temperature-controlled incubation steps were done in a DNA Engine Peltier Thermal Cycler (PTC-200) or DNA Engine OPTICON™from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).