Difference between revisions of "Team:Austin UTexas/Results"

 
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<p style="font-family: verdana">Although bacteria can naturally synthesize GABA, we wanted to <b>increase expression of the <i>gadB</i> gene and subsequently GABA production in order to give our intended probiotic, <i>Lactobacillus plantarum</i>, a more potent medicinal quality</b>, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders, hypertension or anxiety (1). Overexpression of the <i>gadB</i> gene will be accomplished by placing it under the control of either the P8 or P32 constitutive promoters from <i>Lactococcus lactis</i> (2).
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<p style="font-family: verdana">To make our GABA-producing probiotic, we ultimately needed to assemble a GABA overexpression cassette plasmid. The intention is that bacteria containing this GABA overexpression cassette plasmid should produce high levels of GABA. In order to assemble this plasmid, we decided to utilize the Golden Gate Assembly method. In short, Golden Gate Assembly is a relatively new cloning method that allows for the creation of a multi-part DNA assembly (i.e. cassette plasmid) in a single reaction through the use of DNA parts containing specific, predefined suffixes and prefixes with recognition sites for Type IIs restriction enzymes (e.g. BsmBI and BsaI). The specificity of these suffixes and prefixes provides directionality of the desired DNA parts during the assembly process. For our purposes, we used the MoClo Yeast Tool Kit developed by John Dueber (3).</p>
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<p style="font-family: verdana">We decided to first assemble and test our Golden Gate plasmids in <i>E. coli</i>, which was chosen due to the ease in which we could genetically manipulate it. We then wanted to use these Golden Gate plasmids to genetically manipulate <i>L. plantarum</i>. This part of the project required us to assemble a Golden Gate compatible shuttle vector (that is replicable in both <i>E. coli</i> and <i>L. plantarum </i>) and transform <i>L. plantarum</i>.  Our experimental results are detailed below. </p></div>
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<strong><h2 style="font-family: verdana; font-size: 17px; text-align: center; color:  rgb(0, 184, 230)">Click on one of the images below to learn more about our results!</h2></strong>
  
 
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<a href="#section1"><img src="https://static.igem.org/mediawiki/2017/5/5f/Gga-gfp.png" style="width:100%"><p>Part Plasmids </p></a>
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<a href="#section1"><img src="https://static.igem.org/mediawiki/2017/5/5f/Gga-gfp.png" style="width:100%"><p>Part plasmid assembly </p></a>
 
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<a href="#section2"><img src="https://static.igem.org/mediawiki/2017/3/39/Plasmidpart.png" style="width:100%"><p>Cassette Assembly</p></a>
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<a href="#section2"><img src="https://static.igem.org/mediawiki/2017/3/32/Gliw.png" style="width:100%"><p>Testing constitutive <i>Lactococcal</i> Promoters in <i>E. coli</i></p></a>
 
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<a href="#section3"><img src="https://static.igem.org/mediawiki/2017/archive/e/e7/20171101072448%21GABAFinal.png" style="width:100%"> <p>GABA Production </p></a>
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<a href="#section3"><img src="https://static.igem.org/mediawiki/2017/archive/e/e7/20171101072448%21GABAFinal.png" style="width:100%"> <p> Testing <i>gadB</i> Overexpression in <i>E.
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<a href="#section4"><img src="https://static.igem.org/mediawiki/2017/e/e5/Lacto-final.png" style="width:100%"><p><i>Lactobacillus plantarum</i></p></a>
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<a href="#section4"><img src="https://static.igem.org/mediawiki/2017/4/42/Erm.png" style="width:100%"><p>Creating a Golden Gate compatible shuttle vector</p></a>
 
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<strong><h2 style="font-family: verdana; font-size: 17px; text-align: center; color:  rgb(0, 184, 230)">Click on one of the images above to learn more about our results!</h2></strong>
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<a href="#section5"><img src="https://static.igem.org/mediawiki/2017/e/e5/Lacto-final.png" style="width:100%"><p><i>Lactobacillus plantarum</i> transformation</p></a>
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<p style="font-family: verdana">Although bacteria can naturally synthesize GABA, we wanted to <b>increase expression of the <i>gadB</i> gene and subsequently GABA production in order to imbue our probiotic with a more potent medicinal quality</b>, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders or anxiety (1). Overexpression of the <i>gadB</i> gene will be accomplished by placing it under the control of either the P8 or P32 constitutive promoters from <i>Lactococcus lactis</i> (2).
 
  
<p style="font-family: verdana">To make our GABA-producing probiotic we first needed to assemble a GABA overexpression cassette plasmid using the Golden Gate assembly method.</b> The intention here is that bacteria containing this GABA overexpression cassette plasmid should produce high levels of GABA. In short, Golden Gate Assembly is a new cloning method that allows for the creation of a multi-part DNA assembly (i.e. cassette plasmid) in a single reaction through the use of DNA parts containing specific, predefined suffixes and prefixes with recognition sites for Type IIs restriction enzymes (e.g. BsmBI and BsaI). The specificity of these suffixes and prefixes provides directionality of the desired DNA parts during the assembly process. For our purposes, we used the MoClo Yeast Tool Kit developed by John Dueber (3).</p>
 
  
 
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<h2 style="font-family: verdana; font-size: 30px; text-align: center">Part Assembly</h2>
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<p style="font-family: verdana">Additionally, the BsmBI sites and overhangs in pYTK001 are flanking a <i>gfp</i> reporter gene. During the part assembly process, our DNA sequences of interest replaced this <i>gfp</i> reporter gene. This provided a phenotypic screen that allowed us to visually see which transformant colonies were negative and potentially positive. <b>Under UV illumination, positive colonies containing our intended part plasmid assembly did not exhibit fluorescence under the UV illumination, while negative colonies did (Fig. 2)</b>. The non-fluorescent colonies on the part plasmid transformation plates were miniprepped and subsequently sequence verified.</p>
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<p style="font-family: verdana">Additionally, the BsmBI sites and overhangs in pYTK001 are flanking a <i>gfp</i> reporter gene. During the part assembly process, our DNA sequences of interest replaced this <i>gfp</i> reporter gene. This provided a phenotypic screen that allowed us to visually see which transformant colonies were negative and potentially positive. <b>Under UV illumination, positive colonies containing our intended part plasmid assembly did not exhibit fluorescence under the UV illumination, while negative colonies did (Fig. 2)</b>. The non-fluorescent colonies on the part plasmid transformation plates were miniprepped and subsequently sequence verified. Other part plasmids used in subsequent cassette assemblies were directly obtained from UT's Golden Gate Part Repository.</p>
  
 
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<h2 style="font-family: verdana; font-size: 30px; text-align: center">Testing constitutive <i>Lactococcal</i> promoters </h2>
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<p style="font-family: verdana"><b>After successfully creating the <i>gadB</i> gene and P8/P32 promoter part plasmids, the functionality of these part plasmids were then assessed by assembling them into cassette plasmids.</b></p>
 
<p style="font-family: verdana"><b>After successfully creating the <i>gadB</i> gene and P8/P32 promoter part plasmids, the functionality of these part plasmids were then assessed by assembling them into cassette plasmids.</b></p>
  
 
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<p style="font-family: verdana">To test if our <i>Lactococcus lactis</i> constitutive promoters function well within <i>E. coli</i>, we created test cassette plasmids containing the <i>E2-Crimson</i> reporter gene (which encodes a red fluorescent protein) inserted downstream of either the P8 or P32 promoters using BsaI Golden Gate assembly. To create these test cassettes, we used the P8/P32 promoter part plasmids, the <i>E2-Crimson</i> part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone <b>(Fig. 3).</b> <b>If the P8 and P32 promoters are functional in <i>E. coli</i>, we expected to observe red fluorescence in colonies transformed with our test cassette plasmids.</b></p>
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<p style="font-family: verdana">Although the functionality of the <i>Lactococcal lactis</i> P8 and P32 constitutive promoters has been well characterized in Gram-positive bacteria such as <i>Lactobacillus</i>, we wanted to test if these promoters could function well within <i>E. coli</i>. To do this, we created test cassette plasmids containing the <i>E2-Crimson</i> reporter gene (which encodes a red fluorescent protein) inserted downstream of either the P8 or P32 promoters using BsaI Golden Gate assembly. To create these test cassettes, we used the P8/P32 promoter part plasmids, the <i>E2-Crimson</i> part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone <b>(Fig. 3).</b> <b>If the P8 and P32 promoters are functional in <i>E. coli</i>, we expected to observe red fluorescence in colonies transformed with our test cassette plasmids.</b></p>
  
 
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[[File:T--Austin_UTexas--P8P32trans.jpg|thumb|center|800px|<b>Figure 4.</b> P8/P32 test cassette plasmid transformation plates, under normal and UV lights. Under normal lights, the colonies appeared purple-blue in color. Under UV, the colonies fluoresced red.]]
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[[File:T--Austin_UTexas--P8P32trans.jpg|thumb|center|800px|<b>Figure 4.</b> P8/P32 test cassette plasmid transformation plates, under normal (faced up) and UV lights (faced down). Under normal lights, the colonies appeared purple-blue in color. Under UV illumination, the colonies fluoresced red.]]
 
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<h2 style="font-family: verdana; font-size: 30px; text-align: center">Testing for <i>gadB</i> overexpression in <i>E. coli</i></h2>
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<h2 style="font-family: verdana; font-size: 30px; text-align: center">Testing <i>gadB</i> overexpression in <i>E. coli</i></h2>
 
<p style="font-family: verdana"><b>Using Golden Gate Assembly, we created cassette plasmids to test if the <i>gadB</i> gene could be overexpressed in <i>E. coli</i> via the P8 and P32 promoters. </b> These cassette plasmids contained the <i>gadB</i> gene inserted downstream of either the P8 or P32 promoters. To make our <i>gadB</i> test cassette plasmids, we used pYTK095 as our backbone and part plasmids containing the P8/P32 promoters, <i>gadB</i> gene, M13 terminator, and connector sequences <b>(Fig. 5).</b></p>
 
<p style="font-family: verdana"><b>Using Golden Gate Assembly, we created cassette plasmids to test if the <i>gadB</i> gene could be overexpressed in <i>E. coli</i> via the P8 and P32 promoters. </b> These cassette plasmids contained the <i>gadB</i> gene inserted downstream of either the P8 or P32 promoters. To make our <i>gadB</i> test cassette plasmids, we used pYTK095 as our backbone and part plasmids containing the P8/P32 promoters, <i>gadB</i> gene, M13 terminator, and connector sequences <b>(Fig. 5).</b></p>
  
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[[File:GadBmutations.jpg|thumb|center|500 px|<b>Table 1.</b> <i>gadB</i> mutations in sequenced P8/<i>gadB</i> and P32/<i>gadB</i> overexpression cassette plasmids. ]]
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[[File:GadBmutations.jpg|thumb|center|600 px|<b>Table 1.</b> <i>gadB</i> mutations in sequenced P8/<i>gadB</i> and P32/<i>gadB</i> overexpression cassette plasmids. ]]
 
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<p>Along with being one of the canonical amino acids utilized in protein synthesis, glutamate plays an important role as the main amino-group donor in the biosynthesis of nitrogen-containing compounds such as amino acids and nucleotides (4, 5). Thus, we hypothesized that <i>gadB</i> overexpression via the P8 and P32 constitutive promoters and the high-copy-number ColE1 origin induced a high metabolic load on the cells by shunting away glutamate from essential anabolic pathways. Additionally, having high <i>gadB</i> expression does not confer a selective advantage to the cells. We believed that transformants containing the mutationally degraded <i>gadB</i> gene were favored in the population, as "breaking" the metabolically-taxing <i>gadB</i> gene gave these transformants a competitive advantage, allowing them to utilize glutamate sources towards growth. In contrast, transformants containing the functional <i>gadB</i> gene were selected against due to having a depletion of glutamate needed for important cellular processes. </p>
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<p style="font-family: verdana">Along with being one of the canonical amino acids utilized in protein synthesis, glutamate plays an important role as the main amino-group donor in the biosynthesis of nitrogen-containing compounds such as amino acids and nucleotides (4, 5). Thus, we hypothesized that <i>gadB</i> overexpression via the P8 and P32 constitutive promoters and the high-copy-number ColE1 origin induced a high metabolic load on the cells by shunting away glutamate from essential anabolic pathways. Additionally, having high <i>gadB</i> expression does not confer a selective advantage to the cells. We believed that transformants containing the mutationally inactivated <i>gadB</i> gene were favored in the population, as "breaking" the metabolically-taxing <i>gadB</i> gene gave these transformants a competitive advantage, allowing them to utilize glutamate sources towards growth. In contrast, transformants containing the functional <i>gadB</i> gene were selected against due to having a depletion of glutamate needed for important cellular processes. </p>
  
 
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[[File:GadBp15Amutations.jpg|thumb|center|700px|<b>Table 2</b> <i>gadB</i> mutations in sequenced P8/<i>gadB</i>/p15A and P32/<i>gadB</i>/p15A overexpression cassette plasmids.]]
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[[File:GadBp15Amutations.jpg|thumb|center|700px|<b>Table 2.</b> <i>gadB</i> mutations in sequenced P8/<i>gadB</i>/p15A and P32/<i>gadB</i>/p15A overexpression cassette plasmids.]]
 
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<p><b>Given our experimental results and the fact that inducibly expressed genetic devices are more evolutionarily stable than constitutively expressed ones (6), we attempted to inducibly express the <i>gadB</i> gene using the regulatory elements of the lac operon to see if expression and maintenance of a stable <i>gadB</i> gene was possible in <i>E. coli</i></b>. Our IPTG-inducible <i>gadB</i> expression cassette plasmid was assembled using a lac promoter and operator part plasmid, the <i>gadB</i> gene part plasmid, the M13 terminator part plasmid, connector part plasmids, a CP25 promoter part plasmid, a lacI part plasmid, and the SpecR and p15A origin part plasmid <b>(Fig. 8)</b>. Under this assembled regulatory system, in the absence of IPTG (an analog of the allolactose inducer) the LacI repressor will bind to the lac operator region to block transcription of the <i>gadB</i> gene. When present, IPTG will act as an inducer and bind to the LacI repressor to decrease its binding affinity for the lac operator, thereby allowing for <i>gadB</i> expression. This IPTG-inducible system provides us with a mechanism of controlling <i>gadB</i> expression. <b>Positive colonies have been identified and sequence verification is currently underway.</b> </p>
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<p style="font-family: verdana"><b>Given our experimental results and the fact that inducibly expressed genetic devices are more evolutionarily stable than constitutively expressed ones (6), we attempted to inducibly express the <i>gadB</i> gene using the regulatory elements of the <i>lac</i> operon to see if expression and maintenance of a stable <i>gadB</i> gene was possible in <i>E. coli</i></b>. Our IPTG-inducible <i>gadB</i> expression cassette plasmid was assembled using a <i>lac</i> promoter and operator part plasmid, the <i>gadB</i> gene part plasmid, the M13 terminator part plasmid, connector part plasmids, a CP25 promoter part plasmid, a <i>lacI</i> part plasmid, and the SpecR and p15A origin part plasmid <b>(Fig. 8)</b>. Under this assembled regulatory system, in the absence of IPTG (an analog of the allolactose inducer) the LacI repressor will bind to the <i>lac</i> operator region to block transcription of the <i>gadB</i> gene. When present, IPTG will act as an inducer and bind to the LacI repressor to decrease its binding affinity for the <i>lac</i> operator, thereby allowing for <i>gadB</i> expression. This IPTG-inducible system provides us with a mechanism of controlling <i>gadB</i> expression. <b>Positive colonies have been identified and sequence verification is currently underway.</b> </p>
  
  
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<h2 style="font-family: verdana; font-size: 28px; text-align: center">Creating a Golden Gate compatible shuttle vector</h2>
 
<h2 style="font-family: verdana; font-size: 28px; text-align: center">Creating a Golden Gate compatible shuttle vector</h2>
  
<p style="font-family: verdana">We wanted to assemble our final GABA overexpression cassette plasmid using the shuttle vector pMSP3535 as the backbone <b>(Fig. 9).</b> To do this, we first needed to make pMSP3535 Golden Gate compatible (i.e. free of BsaI restriction sites and containing correct overhangs for cassette assembly). We chose to work with pMSP3535 as it contains both a ColE1 origin for replication in <i>E. coli</i> and a pAMb1 origin for replication in Gram-positive bacteria including <i>Lactobacillus</i> species (7). Additionally, the <strong>pMSP3535 vector contains the resistance gene for erythromycin, of which <i>Lactobacillus plantarum</i> is naturally susceptible (8).</strong></p>
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<p style="font-family: verdana">We wanted to assemble our final GABA overexpression cassette plasmid using the shuttle vector pMSP3535 as the backbone <b>(Fig. 9).</b> To do this, we first needed to make pMSP3535 Golden Gate compatible (i.e. free of BsaI restriction sites and containing correct overhangs for cassette assembly). We chose to work with pMSP3535 as it contains both a ColE1 origin for replication in <i>E. coli</i> and a pAMb1 origin for replication in Gram-positive bacteria including <i>Lactobacillus</i> species (7), allowing us to easily transplant our cassette plasmid from <i>E. coli</i> to <i>Lactobacillus plantarum</i> once assembled. Additionally, the <strong>pMSP3535 vector contains the resistance gene for erythromycin, of which <i>Lactobacillus plantarum</i> is naturally susceptible (8).</strong></p>
  
 
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[[File:T--Austin_UTexas--pMSP3535Gel.jpg|thumb|center|700px|Figure 11. (A) Agarose gel of successful PCR for the pMSP3535 backbone and the ColE1 origin. Lane 1 contains a negative PCR control. Lanes 2 and 3 contain the ColE1 origin (around 800 bp) and pMSP3535 backbone PCR products (around 4.3 kb). (B) Agarose gel of diagnostic PCR of miniprep samples from pMSP3535 + ColE1 transformants. Lane 1 contains a negative PCR control. Lane 2 contains a positive PCR control for the pMSP3535 backbone, while lanes 3 and 4 contain the pMSP3535 PCR from two miniprep samples. Lane 7 contains a positive PCR control for the ColE1 origin, while lanes 8 and 9 contain the ColE1 PCR from the same two miniprep samples. Evidently, the pMSP3535 backbone and ColE1 origin is present in both tested miniprep samples.]]
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[[File:T--Austin_UTexas--pMSP3535Gel.jpg|thumb|center|700px|Figure 11. (A) Agarose gel of successful PCR for the pMSP3535 backbone and the ColE1 origin. The ladder used is the 1KB NEB ladder. Lane 1 contains a negative PCR control. Lanes 2 and 3 contain the ColE1 origin (around 800 bp) and pMSP3535 backbone PCR products (around 4.3 kb). (B) Agarose gel of diagnostic PCR of miniprep samples from pMSP3535 + ColE1 transformants. Lane 1 contains a negative PCR control. Lane 2 contains a positive PCR control for the pMSP3535 backbone, while lanes 3 and 4 contain the pMSP3535 PCR from two miniprep samples. Lane 7 contains a positive PCR control for the ColE1 origin, while lanes 8 and 9 contain the ColE1 PCR from the same two miniprep samples. Evidently, the pMSP3535 backbone and ColE1 origin is present in both tested miniprep samples.]]
 
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<p style="font-family: verdana">To this pMSP3535 + ColE1 assembly, we wanted to add a <i>gfp</i> dropout part containing internal BsaI sites that will generate overhangs compatible with those in the P8/P32 promoter and M13 terminator part plasmids.</b> Additionally, the incorporation of this <i>gfp</i> dropout part will also allow us to visually screen for positive and negative transformants based on their fluorescence. BsmBI sites and compatible overhangs were added to the <i>gfp</i> dropout part by PCR amplifying it from pYTK047. We have been attempting to linearize and add BsmBI sites and overhangs to the positive pMSP3535 + ColE1 assemblies via PCR, with no success. However, results from diagnostic digests suggested that our assemblies may have contained extra, undesired DNA such as IS elements <b>(Fig. 12).</b> <b>Thus, as of right now, we are screening for more positive pMSP3535 + ColE1 transformants.</b> Once we have trouble-shooted this problem, the pMSP3535 + ColE1 and the <i>gfp</i> dropout PCR products will be joined through BsmBI assembly to form the final Golden Gate compatible pMSP3535 vector. </p>
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<p style="font-family: verdana">To this pMSP3535 + ColE1 assembly, we wanted to add a <i>gfp</i> dropout part containing internal BsaI sites that will generate overhangs compatible with those in the P8/P32 promoter and M13 terminator part plasmids.</b> Additionally, the incorporation of this <i>gfp</i> dropout part will also allow us to visually screen for positive and negative transformants based on their fluorescence. BsmBI sites and compatible overhangs were added to the <i>gfp</i> dropout part by PCR amplifying it from pYTK047. We have been attempting to linearize and add BsmBI sites and overhangs to the positive pMSP3535 + ColE1 assemblies via PCR, with no success. However, results from diagnostic digests suggested that our assemblies may have contained extra, undesired DNA such as IS elements <b>(Fig. 12).</b> <b>Thus, as of right now, we are screening for more positive pMSP3535 + ColE1 transformants.</b> Once we have troubleshot this problem, the pMSP3535 + ColE1 and the <i>gfp</i> dropout PCR products will be joined through BsmBI assembly to form the final Golden Gate compatible pMSP3535 vector. </p>
  
 
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[[File:T--Austin_UTexas--pMSP3535ColE1Diag.jpg|thumb|center|450px|<b>Figure 12.</b> Agarose gel of pMSP3535 + ColE1 assembly diagnostic digests. Lane 1 contains the undigested plasmid assembly. Lane 2 contains the ClaI-digested plasmid with expected 400 bp and 4.6 kb bands. The actual band generated is apparently above 10 kb. Lane 3 contains the XmnI-digested plasmid with expected 200 bp, 1.5 kb, and 3.3 kb bands. The generated band sizes were 2.5 kb and 4 kb. Lane 4 contains the KpnI-digested assembly with an expected 5.1 kb band. The generated band size was 6 kb. Lane 5 contains the Bg1II-digested assembly with an expected 5.1 kb band. The generated band size was 8 kb.]]
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[[File:T--Austin_UTexas--pMSP3535ColE1Diag.jpg|thumb|center|450px|<b>Figure 12.</b> Agarose gel of pMSP3535 + ColE1 assembly diagnostic digests. The ladder used is the 1KB NEB ladder. Lane 1 contains the undigested plasmid assembly. Lane 2 contains the ClaI-digested plasmid with expected 400 bp and 4.6 kb bands. The actual band generated is apparently above 10 kb. Lane 3 contains the XmnI-digested plasmid with expected 200 bp, 1.5 kb, and 3.3 kb bands. The generated band sizes were 2.5 kb and 4 kb. Lane 4 contains the KpnI-digested assembly with an expected 5.1 kb band. The generated band size was 6 kb. Lane 5 contains the Bg1II-digested assembly with an expected 5.1 kb band. The generated band size was 8 kb.]]
 
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<h3 style="font-family: verdana"; font-size: 28px; text-align: center">Assessing erythromycin susceptibility of <i>E. coli</i></h3>
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<p style="font-family: verdana"><i> Lactobacillus plantarum </i> is a gram-positive lactic acid producing bacteria, so it requires a different growth media than we typically use in our lab. In 1954, Briggs agar was developed (9). This media was designed for <i>Lactobacilli</i>, but was not sufficient for many species, including <i> Lactobacillus plantarum </i>, so a different non-selective media for general lactobacilli was developed in 1960 by Man, Rogosa and Sharpe and named MRS (10). We have exclusively grown our <i> Lactobacillus plantarum </i> on MRS media. Further, we grew <i> Lactobacillus plantarum </i> in a CO2 incubator as referenced in most literature we studied (11-13). The metabolic pathways in the bacteria alter when grown aerobically to produce excess acetate (14) and less lactic acid. Because we intend to utilize this bacteria in a fermentable food, a change in this metabolic pathway would not benefit our ultimate goal.</p>
 
 
 
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<h2 style="font-family: verdana; font-size: 30px; text-align: center"><i>Lactobacillus plantarum</i> transformation</h2>
  <li>MRS broth was prepared using 55g of Difco MRS broth powder in 1L of distilled water. The solution was then autoclaved and stored in a 4 degree refrigerator along with the Difco MRS powder.</li>
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  <li>MRS agar was prepared by mixing 55g of Difco MRS broth powder and 15g of agar flakes in 1 L of water. The solution was then autoclaved and stored in a 4 degree refrigerator along with the Difco MRS powder.</li>
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<p style="font-family: verdana">Once we could successfully grow our chosen bacteria, we needed to  transform the gram positive, <i> Lactobacillus plantarum</i>, with pMSP3535. In order to do this, we identified and worked with a different protocol than we had ever used in our lab. We attempted several protocols, including Landete 2014 (11) and Speer 2012 (12). However, we found success using a variation of the Welker protocol(13). Welker et al.  transformed multiple strains of <i>Lactobacillus casei</i> using varying reagents and yielded different efficiencies between each strain of the species with each variation.</p>
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<p style="font-family: verdana">The Texas Tech iGEM team helped us by testing the Speer protocol after we had attempted three procedures and hadn't successfully transformed. They were not able to successfully transform using the Speer protocol which suggests that the procedure was either too simplified for researchers who have never transformed gram-positive bacteria, or was not compatible with <i>Lactobacillus plantarum.</i>
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  <h4 style="font-family: verdana">Materials Needed</h4>
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  <li>MRS liquid media</li>
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  <li>L. plantarum cells</li>
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  <li>Glass culture tube</li>
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  <li>CO2 incubator</li>
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  <li>MRS liquid media, containing 0.5 M sucrose</li>
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  <li>Spectrophotometer</li>
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  <li>Centrifuge</li>
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  <li>Vortex</li>
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  <li>50 mL Falcon conical tubes (2)</li>
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  <li>30% PEG 8000</li>
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  <h4>Preparation of Recipient Cell Stocks</h4>
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  <li>Inoculate 10-25 mL of -80 &#2103 L. Plantarum stocks in MRS broth at 37 &#2103 CO2 incubator, overnight without shaking.</li>
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  <li>Subculture by incubating culture in a 200 mL of prewarmed MRS broth. Add culture until OD600 is 0.1.</li>
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  <li>Subcultures should be grown to an OD600 of 0.6 (4-8 hours).</li>
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  <li>For glycine supplementation, the MRS broth should contain 0.5%-1.0% glycine.</li>
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  <li>For NaCl supplementation, inoculate subcultures with 0.9M of NaCl.</li>
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  <li>Harvest cells from the subculture by centrifugation at 4 &#2103 for 10 min. at 7000 rpm after inoculation.</li>
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  <li>Rinse the cells in 200 mL of sterile, distilled water (~4 &#2103) and centrifuge again.</li>
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  <li>Resuspend the pellet in 2-3 mL of cold, sterile, distilled water and aliquot 1 mL volumes to 1.5 mL microcentrifuge tubes.</li>
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  <li>Centrifuge aliquots at 15,000 rpm for ~90 seconds and remove the supernatant.</li>
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  <li>Rinse the cells twice with 1 mL of cold, distilled, sterile water. Then rinse with 1 mL of cold, sterile 30% PEG-8000 solution. Remove the supernatant.</li>
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  <li>Suspend cells in 0.5-0.6 mL of 30% PEG for storage at -80 &#2103. These should retain viability for up to 2 years.</li>
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  <h4>Treatment of Cells Prior to Electroporation</h4>
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  <li>Thaw on Ice.</li>
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  <li>Suspend cells in  900μL of cold, sterile, distilled water for 30 min.</li>
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  <li>Treat with 100mM lithium acetate and 10mM DTT solution for 30min.</li>
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  <li>After pretreatment, the cells were pelleted for 2-3 minutes in a microcentrifuge, washed once in 1 mL of cold, sterile, 30% PEG solution and suspended in 0.5-0.6 mL cold, sterile PEG solution for electroporation.</li>
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  <li>Isolate intended vector (Lactobacillus plantarum experiment as of 09/06/17 has been using pMSP3535).</li>
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  <li>200 ng/transformation of plasmid DNA is mixed with 100μL of cell suspension and transferred to a prechilled electroporation cuvette with a 0.2 cm gap.</li>
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  <li>Pulse at 12.5kV (From Literature: Bio-rad gene pulser with capacitance setting at 25μF and resistances of 200 or 400 Ω and voltage settings of 1500, 2000 or 2500 V (7.5, 10 or 12.5 kV cm-1).</li>
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  <li>Quickly recover cells by adding 900μL of recovery medium (0.5 M sucrose in MRS broth) to the cuvette then transfer the cells to a sterile microcentrifuge tube for incubation ~4 hours at 37oC in a CO2 incubator without shaking. (*Save recovery and plate again if no growth)</li>
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  <li>Dilute cells in recovery medium and plate on MRS agar along with the proper concentration of any antibiotics that the plasmid of choice has resistancy (pMSP3535 has resistance to erythromycin).</li>
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  <li>Incubate plates for 2 days, then count the colonies present. Allow an additional 3-4 days after initial count to ensure all were noted.</li>
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  <li>Cells stored in glycerol (-80 &#2103) after electroporation incubation can be expected to yield similar results to the cells that were plated directly after the incubation.</li>
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<p style="font-family: verdana">After preparing the necessary solutions, we followed the Welker protocol with some minor differences. We inoculated bacterial stocks with 10 mL of MRS broth in a CO2 incubator, without shaking, overnight. After this, we subcultured the bacteria in 200 mL of prewarmed MRS broth with 0.9M NaCl from an OD600 of 0.1 until it reached an OD600 of 0.6 (~6 hours). After harvesting the cells by centrifugation and rinsing, we resuspended the cells in 4 mL of cold water and eight 0.5 mL aliquots were divided into 1.5 mL microcentrifuge tubes.</p>
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<p style="font-family: verdana"> In order to grow <i>Lactobacillus plantarum</i>, we used MRS broth in a CO<sub>2</sub> incubator without shaking. After characterizing the growth of the bacteria, we aimed to transform it with pMSP3535. We first attempted several protocols, including Landete 2014 (11) and Speer 2012 (12). However, we found success using a variation of the Welker protocol (13). After preparing the necessary solutions, we followed the Welker protocol with some minor differences, including inoculation of bacterial stocks in 10 mL of MRS broth rather than 25mL; utilizing a CO<sub>2</sub> incubator without shaking; subculturing cells in 0.9M NaCl rather than glycine, transforming with 100 ng of plasmid DNA  instead of the 200 ng that the protocol recommends, and electroporating with a resistance of 3kV and 600Ω. For more information about the protocols including broth recipe and transformation procedure, click <a href="https://2017.igem.org/Team:Austin_UTexas/Protocols">here!</a></p>
  
 
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<p style="font-family: verdana">The cells were washed several more times with water and 30% PEG 8000, then stored in a -80°C freezer. These cells should stay viable for up to two years.To enhance the transformation efficiency, 600 μL of prepared cells were suspended 900 μL of cold, sterile, distilled water for 30 min as a pretreatment to electroporation.The cells were pelleted and washed several times with water and 30% PEG 8000. We added 100 ng of plasmid DNA to 100μL of cell suspension as opposed to 200 ng that the protocol recommends; our first plasmid of choice was pMSP3535. This plasmid expresses erythromycin resistance. The cells were electroporated in 2mm cuvettes using the Ec3 settings on the BioRad electroporator which corresponds to 600Ω and 3 kV, differing from the Welker protocol recommendations.
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<p style="font-family: verdana"> Our first plasmid we chose to transform <i> Lactobacillus plantarum </i> with was pMSP3535. This plasmid expresses erythromycin resistance and contains the pAMB1 origin.  
After cells were electroporated, they recovered overnight in the appropriate recovery media. They were plated on MRS agar plates with 10μg/mL erythromycin and left to grow for 2 days. </p>
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After cells were electroporated, they recovered overnight in the appropriate recovery media. They were plated on MRS agar plates supplemented with 10 μg/mL erythromycin and left to grow for 2 days. The results of our transformation can be seen in <b>Figure 14</b>.</p>
  
 
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[[File:T--Austin_UTexas--LPlantarumTrans.jpg|thumb|center|800px]]
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[[File:Orglacto.png|thumb|center|450px|<strong>Figure 14.</strong> Transformations of <i>Lactobacillus plantarum</i> with pMSP3535. Plate A corresponds to the negative control: electroporated cells at 3kV and 600 Ω with no plasmid DNA. Plate B corresponds to cells with 100 ng of pMSP3535 plasmid DNA electroporated at 2kV and 400 Ω. Plate  cells were electroporated at 2.5kV and 400 Ω with 100 ng of pMSP3535 plasmid DNA. Plate D corresponds to the cells electroporated at 3kV and 600 Ω with 100 ng of pMSP3535.]]
 
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<p style="font-family: verdana">Colonies from each transformation plate were grown up in MRS broth supplemented with 10μg/mL erythromycin. The subsequent day, they were streaked on  10μg/mL erythromycin MRS agar plates to verify resistance.</p>
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<p style="font-family: verdana">Colonies from each transformation plate were grown up in MRS broth supplemented with 10 μg/mL erythromycin. The next day, they were streaked on  10μg/mL erythromycin MRS agar plates to verify resistance to erythromycin. The overnight cultures and re-streaks are shown in <b>Figure 15</b> and <b>Figure 16</b> respectively.</p>
  
 
<strong><p style="font-family: verdana"> Genomic and plasmid sequence verification is underway.</p></strong>
 
<strong><p style="font-family: verdana"> Genomic and plasmid sequence verification is underway.</p></strong>
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[[File:T--Austin_UTexas--LPlantarumExp.jpg|thumb|center|800px]]
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[[File:Tubes.png|thumb|center|350px|'''<b>Figure 15.</b>'''Overnight cultures of transformed<i>Lactobacillus plantarum</i> were grown in erythromycin. Tube A is the negative control: untransformed <i> Lactobacillus plantarum</i> in MRS broth supplemented with 10μg/mL erythromycin. Tube B was our positive control: transformed <i> Lactobacillus plantarum</i> in MRS broth. Tube C is the experimental cells: transformed <i>Lactobacillus plantarum</i> grown in MRS broth supplemented in 10μg/mL erythromycin.'']]
 
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[[File:Restreaks.png|thumb|center|350px|'''<b>Figure 16.</b>''' This figure displays a transformed <i>Lactobacillus plantarum</i> re-streak plated on MRS agar supplemented with 10μg/mL erythromycin beside a negative control. Plate A corresponds to untransformed <i>L. plantarum</i> on the selective media and Plate B corresponds to the transformed bacteria on selective media.'']]
 
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<p style="font-family: verdana">We aimed to show a visible transformation of <i>Lactobacillus plantarum</i>  in addition to the resistance, so we transformed again using a different plasmid. This time we transformed with  pBTK501, a plasmid that codes for a resistance to ampicillin and expresses <i>gfp</i>.</p>
 
  
 
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<li>Flórez A. B. et al. Susceptibility of <i>Lactobacillus plantarum</i> Strains to Six Antibiotics and Definition of New Susceptibility–Resistance Cutoff Values. <i>Microbial Drug Resistance</i>. 12(4): 252-56 (2007).</li>
 
<li>Flórez A. B. et al. Susceptibility of <i>Lactobacillus plantarum</i> Strains to Six Antibiotics and Definition of New Susceptibility–Resistance Cutoff Values. <i>Microbial Drug Resistance</i>. 12(4): 252-56 (2007).</li>
  
<li> Cox, C. P., & Briggs, M. (1954). Experiments On Growth Media For Lactobacilli. Journal of Applied Bacteriology, 17(1), 18-26. doi:10.1111/j.1365-2672.1954.tb02019.x</li>
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<li> Cox, C. P., & Briggs, M. (1954). Experiments On Growth Media For <i>Lactobacilli</i>. Journal of Applied Bacteriology, 17(1), 18-26. doi:10.1111/j.1365-2672.1954.tb02019.x</li>
  
<li>Man, J. C., Rogosa, M., & Sharpe, M. E. (1960). A Medium For The Cultivation Of Lactobacilli. Journal of Applied Bacteriology, 23(1), 130-135. doi:10.1111/j.1365-2672.1960.tb00188.x</li>
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<li>Man, J. C., Rogosa, M., & Sharpe, M. E. (1960). A Medium For The Cultivation Of <i>Lactobacilli</i>. Journal of Applied Bacteriology, 23(1), 130-135. doi:10.1111/j.1365-2672.1960.tb00188.x</li>
  
<li>Speer, M., & Richard, T. (n.d.). Lactobacillus transformation (Speer 2012). Retrieved May 15, 2017, from https://openwetware.org/wiki/Lactobacillus_transformation_(Speer_2012)</li>
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<li>Speer, M., & Richard, T. (n.d.).<i> Lactobacillus</i> transformation (Speer 2012). Retrieved May 15, 2017, from https://openwetware.org/wiki/Lactobacillus_transformation_(Speer_2012)</li>
  
 
<li>Landete, J. M., Arqués, J. L., Peirotén, Á, Langa, S., & Medina, M. (2014). An improved method for the electrotransformation of lactic acid bacteria: A comparative survey. Journal of Microbiological Methods, 105, 130-133. doi:10.1016/j.mimet.2014.07.022</li>
 
<li>Landete, J. M., Arqués, J. L., Peirotén, Á, Langa, S., & Medina, M. (2014). An improved method for the electrotransformation of lactic acid bacteria: A comparative survey. Journal of Microbiological Methods, 105, 130-133. doi:10.1016/j.mimet.2014.07.022</li>
  
<li>Dennis L. Welker, Joanne E. Hughes, James L. Steele, Jeff R. Broadbent; High efficiency electrotransformation of Lactobacillus casei, FEMS Microbiology Letters, Volume 362, Issue 2, 1 January 2015, Pages 1–6, https://doi.org/10.1093/femsle/fnu033</li>
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<li>Dennis L. Welker, Joanne E. Hughes, James L. Steele, Jeff R. Broadbent; High-efficiency electrotransformation of <i>Lactobacillus casei</i>, FEMS Microbiology Letters, Volume 362, Issue 2, 1 January 2015, Pages 1–6, https://doi.org/10.1093/femsle/fnu033</li>
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<li>Murphy, M. G., & Condon, S. (1984). Comparison of aerobic and anaerobic growth of <i>Lactobacillus plantarum</i> in a glucose medium. Archives of Microbiology, 138(1), 49-53. </li>
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<li>GABA 3D ball-and-stick model, viewed 1 November 2017, by Jynto, from <a href="https://commons.wikimedia.org/wiki/File:GABA_3D_ball.png#file">Wikimedia Commons.</a></li>
  
<li>Murphy, M. G., & Condon, S. (1984). Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium. Archives of Microbiology, 138(1), 49-53. </li>
 
  
 
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Latest revision as of 07:00, 21 November 2017


Results

Although bacteria can naturally synthesize GABA, we wanted to increase expression of the gadB gene and subsequently GABA production in order to give our intended probiotic, Lactobacillus plantarum, a more potent medicinal quality, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders, hypertension or anxiety (1). Overexpression of the gadB gene will be accomplished by placing it under the control of either the P8 or P32 constitutive promoters from Lactococcus lactis (2).

To make our GABA-producing probiotic, we ultimately needed to assemble a GABA overexpression cassette plasmid. The intention is that bacteria containing this GABA overexpression cassette plasmid should produce high levels of GABA. In order to assemble this plasmid, we decided to utilize the Golden Gate Assembly method. In short, Golden Gate Assembly is a relatively new cloning method that allows for the creation of a multi-part DNA assembly (i.e. cassette plasmid) in a single reaction through the use of DNA parts containing specific, predefined suffixes and prefixes with recognition sites for Type IIs restriction enzymes (e.g. BsmBI and BsaI). The specificity of these suffixes and prefixes provides directionality of the desired DNA parts during the assembly process. For our purposes, we used the MoClo Yeast Tool Kit developed by John Dueber (3).

We decided to first assemble and test our Golden Gate plasmids in E. coli, which was chosen due to the ease in which we could genetically manipulate it. We then wanted to use these Golden Gate plasmids to genetically manipulate L. plantarum. This part of the project required us to assemble a Golden Gate compatible shuttle vector (that is replicable in both E. coli and L. plantarum ) and transform L. plantarum. Our experimental results are detailed below.


Click on one of the images below to learn more about our results!