Line 253: | Line 253: | ||
<br> | <br> | ||
− | <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 (Fig. 12). | + | <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 (Fig. 12). Thus, as of right now, we are screening for more positive pMSP3535 + ColE1 transformants. 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. |
<br> | <br> | ||
Line 267: | Line 267: | ||
<br> | <br> | ||
− | <p style="font-family: verdana"> | + | <p style="font-family: verdana">Because we are creating our Golden Gate compatible pMSP3535 shuttle vector in <i>E. coli</i>, we wanted to determine the natural susceptibility of <i>E. coli</i> to erythromycin as the minimum concentration to use has not been established clearly in the literature. Thus, we performed an erythromycin minimum inhibitory concentration test in liquid LB media <b>(Fig. 13).</b> After one-day incubation, we observed that <i>E. coli</i> was resistant up to around 150 µg/mL of erythromycin. <b>From this experiment, we have determined that the optimal erythromycin concentration for selecting against <i>E. coli</i> in liquid culture is around 200-250 µg/mL.</b></p> |
<br> | <br> |
Revision as of 06:34, 1 November 2017
Click on one of the images below to learn more about our results!
Although bacteria can naturally synthesize GABA, we wanted to increase expression of the gadB gene and subsequently GABA production in order to imbue our probiotic with a more potent medicinal quality, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders 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 first needed to assemble a GABA overexpression cassette plasmid using the Golden Gate assembly method. 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).
Creating a Golden Gate compatible shuttle vector
We wanted to assemble our final GABA overexpression cassette plasmid using the shuttle vector pMSP3535 as the backbone (Fig. 9). 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 E. coli and a pAMb1 origin for replication in Gram-positive bacteria including Lactobacillus species (6). Additionally, the pMSP3535 vector contains the resistance gene for erythromycin, which Lactobacillus plantarum is naturally susceptible to (7).
The process of making the pMSP3535 vector Golden Gate compatible involved two steps: 1) assembling the pMSP3535 backbone (pAMb1 origin and erythromycin resistance gene) with a new ColE1 origin; 2) assembling a gfp dropout part to the assembly of the pMSP3535 backbone and the new ColE1 origin (Fig. 10).
Assessing erythromycin susceptibility of E. coli
Because we are creating our Golden Gate compatible pMSP3535 shuttle vector in E. coli, we wanted to determine the natural susceptibility of E. coli to erythromycin as the minimum concentration to use has not been established clearly in the literature. Thus, we performed an erythromycin minimum inhibitory concentration test in liquid LB media (Fig. 13). After one-day incubation, we observed that E. coli was resistant up to around 150 µg/mL of erythromycin. From this experiment, we have determined that the optimal erythromycin concentration for selecting against E. coli in liquid culture is around 200-250 µg/mL.