Team:Utah State/Broad-HostVectors

Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 E. coli cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent E. coli and tested for successful insertion using PCR and restriction digests.

Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.

In order to transfer a vector of interest using conjugation, the tra gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:

• Hfr strain – The tra operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.
• Biparental (normal) Conjugation – Cells containing the tra genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell
• Triparental Mating – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.

For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.

Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.

Table 2 Components and selection criteria used in conjugation with the broad-host vector PRL1383A

Results

Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.

In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.

Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid.

Figure 4 Results of the tri-parental mating between pCPP33 and R. sphaeroides, P. putida, and Synechocystis sp., respectively. Each plate is shown alongside a negative control