Team:Utah State/Broad-HostVectors

From 2009.igem.org

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<p class="class">The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in <i>E. coli</i>. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).</p>
<p class="class">The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in <i>E. coli</i>. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).</p>
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Experimental Section: Approach for BioBrick Compatibility
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Converting Broad-Host Vectors into a BioBrick-Compatible Format
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<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg""  align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div>
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<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20>
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<b>Figure 2</b>  Plasmid map of pRL1383a
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/PCPP33_Plasmid_Map.jpg""  align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div>
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<b>Figure 3</b>  Plasmid map of pCPP33
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<p class="class">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 <i>E. coli</i> 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 <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p>
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<p class="class">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.</p>
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Broad Host Conjugation
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<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> 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:</p>
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</li><li><b>Hfr strain</b> – The <i>tra</i> 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.
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</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> 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
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</li><li><b>Triparental Mating</b> – 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.
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<p class="class">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.</p>
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<p class="class">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.</p>
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png"  align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div>
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<b>Table 1</b>  Components and selection criteria used in conjugation with the broad-host vector PCPP33
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png"  align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div>
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<b>Table 2</b>  Components and selection criteria used in conjugation with the broad-host vector PRL1383A
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Results
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<p class="class">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.</p>
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<p class="class">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.</p>
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<p class="class">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.
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG"  align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG"  align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG"  align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div>
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<b>Figure 4</b>  Results of the tri-parental mating between pCPP33 and <i>R. sphaeroides</I>, <i>P. putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control
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Revision as of 03:20, 22 October 2009

USU iGem

Untitled Document

PROJECT Abstract
Introduction
Broad-Host Vectors
Secretion Experiments Future Work References
Broad-Host Range Vectors

Advantages for Using a Broad Host Range Vector

A multi-host vector allows for genetic manipulation to occur in one organism, and the ultimate application of the vector to be served in another. Genetic manipulation is ideally done in E. coli, due to its fast growth, ease of use, and availability of transformable cells. However, it does not always represent the best choice for production of recombinant proteins or other compounds, and thus it is ideal to be able to transfer genetic information into other organisms once manipulation and testing of the construct is complete.

Most broad host range vectors are naturally occurring or a derivative of a natural vector. They tend to be large, around 10 kbp, although some commercial versions have been optimized to a much shorter length (http://www.bio101.com/functional-analysis/pBBR122.html). They can be self-transmissible (presence of tra genes) and mobilizable (mob genes), but desirable vectors are both mobilizable and non-transmissible (Haller, & Dichristina, 2002). This allows for more control over conjugation in the laboratory through use of a helper plasmid (Haller, & Dichristina, 2002). A helper plasmid is a conjugative plasmid, that is it contains both transmission and mobilization genes. While a broad-host range plasmid can be conjugated into another organism, its copy number will remain undetectably low unless a fully functioning helper plasmid is present (Haller, & Dichristina, 2002).. If a helper plasmid shares the same origin of transfer (oriT), mob genes are no longer necessary (Snyder and Champness 2007). Due to this property, the mob genes of commercial plasmids are often removed, thereby resulting in vectors that are significantly shorter than their natural counterparts (Snyder and Champness 2007). Use of a helper plasmid becomes necessary if the self-transmission genes are not present to achieve any detectable degree of replication in the recipient organism (Haller, & Dichristina, 2002).

Genetic Characteristics of Broad-Host Range Vectors

Broad host range vectors are a class of mobilizable plasmids, that is they lack the complete tra-genes necessary for conjugation but can still transfer and replicate at high copy number in the presence of a conjugative plasmid. Mobilizable vectors still contain some of the genes necessary for transfer. The mob genes code proteins that aid the vector in transferring from one organism to another. One protein produced in the region, nickase-helicase, nics the DNA at the origin of transfer (oriT). As the envelopes of the two cells meet, the mobility proteins synthesize a new strand of DNA from the plasmid parent strand as it enters the recipient cell. A new strand is also synthesized in the donor cell simultaneously. In this way, the plasmid is transferred from one cell to another (Porter, 2002).


Team USU
Figure 1. Mechanism for bacterial conjugation

The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in E. coli. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).



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