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

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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!

Part plasmid assembly

Testing constitutive Lactococcal Promoters in E. coli

Testing gadB Overexpression in E. coli

Creating a Golden Gate compatible shuttle vector

Lactobacillus plantarum transformation

Part plasmid assembly

The first part of our Golden Gate assembly workflow was part assembly, in which the gadB gene and the P8/P32 promoters were individually cloned into the entry vector pYTK001 (Fig. 1). The gadB gene and P8/P32 promoter sequences contain flanking BsmBI sites that produce overhangs compatible with those cut by BsmBI in the entry vector pYTK001. Thus, BsmBI cloning should result in part plasmids containing the gadB gene and P8/P32 promoters set within the pYTK001 backbone.

Figure 1. gadB gene and P8/P32 promoter part assembly process. Golden Gate compatible gadB and P8/P32 promoter sequences are cloned into the pYTK001 entry vector via BsmBI assembly.

Additionally, the BsmBI sites and overhangs in pYTK001 are flanking a gfp reporter gene. During the part assembly process, our DNA sequences of interest replaced this gfp reporter gene. This provided a phenotypic screen that allowed us to visually see which transformant colonies were negative and potentially positive. 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). 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.

Figure 2. gadB gene and P8/P32 promoter part plasmid E. coli transformations, compared to control transformations with pYTK001. Under UV illumination, transformants containing the correctly assembled part plasmids were non-fluorescent while negative transformants appeared fluorescent like colonies on the control plates.

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Testing constitutive Lactococcal promoters in E. coli

After successfully creating the gadB gene and P8/P32 promoter part plasmids, the functionality of these part plasmids were then assessed by assembling them into cassette plasmids.

Although the functionality of the Lactococcal lactis P8 and P32 constitutive promoters has been well characterized in Gram-positive bacteria such as Lactobacillus, we wanted to test if these promoters could function well within E. coli. To do this, we created test cassette plasmids containing the E2-Crimson 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 E2-Crimson part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone (Fig. 3). If the P8 and P32 promoters are functional in E. coli, we expected to observe red fluorescence in colonies transformed with our test cassette plasmids.

Figure 3. Golden Gate assembly process of the P8 and P32 test cassette plasmids.

Upon first look, E. coli colonies transformed with these assemblies appeared purple-blue in color. This phenotype was due to the expression of the red fluorescent protein. Further, we noticed that E. coli colonies transformed with these assemblies fluoresced red under UV light, indicating that the P8 and P32 promoters are indeed expressing the E2-Crimson reporter gene and thus are functional in E. coli (Fig. 4).

Figure 4. 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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Testing gadB overexpression in E. coli

Using Golden Gate Assembly, we created cassette plasmids to test if the gadB gene could be overexpressed in E. coli via the P8 and P32 promoters. These cassette plasmids contained the gadB gene inserted downstream of either the P8 or P32 promoters. To make our gadB test cassette plasmids, we used pYTK095 as our backbone and part plasmids containing the P8/P32 promoters, gadB gene, M13 terminator, and connector sequences (Fig. 5).

Figure 5. Golden Gate assembly process of P8/gadB and P32/gadB test cassette plasmids.

Similar to the pYTK001 entry vector in part assembly, the pYTK095 backbone used for cassette assembly contained a gfp reporter gene that is replaced by sequences of interest. This allowed us to easily perform a phenotypic screen for positive colonies. Non-fluorescent colonies may potentially have had the correct cassette assembly, while fluorescent colonies did not (Fig. 6).

Figure 6. P8/gadB and P32/gadB cassette E. coli transformations alongside a pYTK095 control transformation under UV illumination. Potentially positive colonies containing the correct assemblies appeared non-fluorescent, while negative colonies appeared fluorescent.

The non-fluorescent colonies were then screened using colony PCR, and positive colonies were then miniprepped and sequenced. The sequencing results indicated that there were several mutations within the gadB gene in the samples. These mutations are recorded in Table 1.

Table 1. gadB mutations in sequenced P8/gadB and P32/gadB overexpression cassette plasmids.

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 gadB 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 gadB expression does not confer a selective advantage to the cells. We believed that transformants containing the mutationally inactivated gadB gene were favored in the population, as "breaking" the metabolically-taxing gadB gene gave these transformants a competitive advantage, allowing them to utilize glutamate sources towards growth. In contrast, transformants containing the functional gadB gene were selected against due to having a depletion of glutamate needed for important cellular processes.

To troubleshoot this problem, we decided to use a backbone containing the low-copy number p15A origin for the cassette assembly (Fig. 7).

Figure 7. Golden Gate assembly process of P8/gadB and P32/gadB test cassette plasmids using a backbone containing the low-copy number p15A origin.

Copy number reduction of our gadB overexpression cassette plasmids (accomplished by replacing the ColE1 origin with the p15A origin) was expected to decrease the overall amount of gadB expression, thereby decreasing the metabolic load imposed on the cells and the mutation rates observed in the gadB gene. However, we also observed several mutations within the gadB gene in the potentially positive cassette plasmids sequenced. The observed mutations are detailed in Table 2.

Table 2. gadB mutations in sequenced P8/gadB/p15A and P32/gadB/p15A overexpression cassette plasmids.

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 gadB gene using the regulatory elements of the lac operon to see if expression and maintenance of a stable gadB gene was possible in E. coli. Our IPTG-inducible gadB expression cassette plasmid was assembled using a lac promoter and operator part plasmid, the gadB 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 (Fig. 8). 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 gadB 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 gadB expression. This IPTG-inducible system provides us with a mechanism of controlling gadB expression. Positive colonies have been identified and sequence verification is currently underway.

Figure 8. Golden Gate assembly process of IPTG-inducible gadB expression cassette plasmid.

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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 (7), allowing us to easily transplant our cassette plasmid from E. coli to Lactobacillus plantarum once assembled. Additionally, the pMSP3535 vector contains the resistance gene for erythromycin, of which Lactobacillus plantarum is naturally susceptible (8).

Figure 9. Golden Gate assembly of the GABA final overexpression cassette plasmid with the Golden Gate compatible pMSP3535 vector and the P8/P32 promoter, gadB gene, and M13 terminator part plasmids.

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).

Figure 10. Assembly workflow for creating the Golden Gate compatible pMSP3535 backbone. The first step involves replacing the ColE1 origin in the pMSP3535 backbone with a new ColE1 origin from pYTK001 to create a pMSP3535 + ColE1 assembly. The second step involves combining the pMSP3535 + ColE1 assembly with a gfp dropout to form the final Golden Gate compatible vector to be used to create our GABA overexpression plasmid.

Since the original pMSP3535 vector contained two illegal BsaI sites within the ColE1 origin, we sought to replace this ColE1 origin with a BsaI-free one isolated from pYTK001. This assembly process involved linearizing and adding BsmBI sites and compatible overhangs to the pMSP3535 backbone and the pYTK001 ColE1 origin via PCR. After the pMSP3535 backbone and ColE1 origin were successfully amplified by PCR (Fig. 11a), they were joined together using BsmBI assembly. Diagnostic PCR was performed on pMSP3535 + ColE1 minipreps from E. coli transformants to screen for positive samples containing both the pMSP3535 backbone and the ColE1 inserts (Fig. 11b). After confirming the presence of the pMSP3535 vector and ColE1 origin, we partially sequence-confirmed the two miniprep samples.

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.

To this pMSP3535 + ColE1 assembly, we wanted to add a gfp dropout part containing internal BsaI sites that will generate overhangs compatible with those in the P8/P32 promoter and M13 terminator part plasmids. Additionally, the incorporation of this gfp 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 gfp 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 troubleshot this problem, the pMSP3535 + ColE1 and the gfp dropout PCR products will be joined through BsmBI assembly to form the final Golden Gate compatible pMSP3535 vector.

Figure 12. 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.

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.

Figure 13. Erythromycin minimum inhibitory concentration tests for E. coli in liquid media. From left to right: 0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL, 350 µg/mL, and 400 µg/mL.

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Lactobacillus plantarum transformation

In order to grow Lactobacillus plantarum, we used MRS broth in a CO₂ 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 CO₂ 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 here!

Our first plasmid we chose to transform Lactobacillus plantarum 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 supplemented with 10 μg/mL erythromycin and left to grow for 2 days. The results of our transformation can be seen in Figure 14.

Figure 14. Transformations of Lactobacillus plantarum 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.

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 Figure 15 and Figure 16 respectively.

Genomic and plasmid sequence verification is underway.

Figure 15.Overnight cultures of transformedLactobacillus plantarum were grown in erythromycin. Tube A is the negative control: untransformed Lactobacillus plantarum in MRS broth supplemented with 10μg/mL erythromycin. Tube B was our positive control: transformed Lactobacillus plantarum in MRS broth. Tube C is the experimental cells: transformed Lactobacillus plantarum grown in MRS broth supplemented in 10μg/mL erythromycin.

Figure 16. This figure displays a transformed Lactobacillus plantarum re-streak plated on MRS agar supplemented with 10μg/mL erythromycin beside a negative control. Plate A corresponds to untransformed L. plantarum on the selective media and Plate B corresponds to the transformed bacteria on selective media.

References

Yunes, R. A et al. GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota. Anaerobe. 42: 197-204 (2016).
Zhu, D. et al. Isolation of strong constitutive promoters from Lactococcus lactis subsp. Lactis N8. FEMS Microbiol Lett. 363(16): pii: fnv107 (2015).
Lee, M. E. et al. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4(9): 975-86 (2015).
Commichau, F. M., et al. Glutamate metabolism in Bacillus subtilis: Gene Expression and Enzyme Activities Evolved To Avoid Futile Cycles and to allow Rapid Responses to Perturbations of the System. AMS. 190(10): 3557-64 (2008).
Sarkar, P.K. and Nout, M.J.R. (Eds.) 2014. Handbook of Indigenous Foods Involving Alkaline Fermentation. CRC Press/Taylor & Francis Group, p. 629.
Sleight, S. C. et al. Designing and engineering evolutionary robust genetic circuits. Journal of Biological Engineering. (4):12 (2010).
Pérez-Arellano, I. et al. Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia coli. Plasmid. 46(2): 106-16 (2001).
Flórez A. B. et al. Susceptibility of Lactobacillus plantarum Strains to Six Antibiotics and Definition of New Susceptibility–Resistance Cutoff Values. Microbial Drug Resistance. 12(4): 252-56 (2007).
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
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
Speer, M., & Richard, T. (n.d.). Lactobacillus transformation (Speer 2012). Retrieved May 15, 2017, from https://openwetware.org/wiki/Lactobacillus_transformation_(Speer_2012)
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
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
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.
GABA 3D ball-and-stick model, viewed 1 November 2017, by Jynto, from Wikimedia Commons.

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-<p> This year our team created a mathematical representation of our Genetically Engineered Artificial Ratio (G.E.A.R.) system. This representation, or model, informed the wet lab on the timescales of the three modules of GEAR: communication, comparator and growth regulator. It also helped develop assembly strategies thanks to a thorough analysis of parameter scans and sensitivities. After a series of experiments in the wet lab, we were able to finesse our parameters making them more accurate. This process, presented below, can be seen as a continuous feedback between the wet lab and the dry lab of Ecolibrium.<br><br><br></p>
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-We developed our models with two main goals in mind:
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-<li>We wanted our model to be able to aid the team in the wetlab. Especially for optimizing the assembly process and balancing the circuit.</li>
-<li>We wanted to develop an in silico version of our circuit at single cell and population level. This allowed us to test the viability of the system in both normal and abnormal conditions. It also allows us to plan future experiments and used for the system.</li>
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-The following pages will show how we implemented modelling approaches to achieve our goals.
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-<li>Communication Module - We constructed the four quorum systems that we considered viable choices for our system (cin, rhl, lux and las)to allow us to directly compare the expected behaviour and plan our growth module experiments accordingly.
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-Population Model </a></specialh4><br>
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-<li> Matlab population model with GP2 growth regulation in two population
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-<li>We used the GRO programming language to model a simplified version of our circuit with GP0.4 growth regulation into two populations of E.Coli.
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+<div class="clear"></div>
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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).
-<div class="col-lg-10 col-centered">
+<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>
-<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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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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-                    <div class="col-md-11">Species</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+<br>
+<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="#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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+</div>
-<specialh4> Table 1: Universal Species Table </specialh4>
+<div class="column fifth_size">
-<table class="table table-bordered table-hover">
+<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.
-<thead>
+  coli</i></p></a>
-        <tr>
+</div>
-            <th>Species Name</th>
-            <th>Description</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>C4</td>
-            <td>AHLs produced by RhlI synthase </td>
-        </tr>
-        <tr>
-            <td>C14</td>
-            <td>AHLs produced by CinI synthase</td>
-        </tr>
-        <tr>
-            <td>g_C4R</td>
-            <td>Copy number for plasmid backbone housing C4R </td>
-        </tr>
-        <tr>
-            <td>mC4R</td>
-            <td>mRNA for RhlR</td>
-        </tr>
-        <tr>
-            <td>C4R</td>
-            <td>RhlR transcriptional regulator  </td>
-        </tr>
-        <tr>
-            <td>C4Comp</td>
-            <td>Complex by C4 AHL and RhlR transcriptional regulator </td>
-        </tr>
-        <tr>
-            <td>g_STARC4Comp</td>
-            <td>Complex formed between plasmid backbone for STAR (pRhl) and c4 Comp  </td>
-        </tr>
-        <tr>
-            <td>g_C14R</td>
-            <td>Copy number for plasmid backbone housing C14R </td>
-        </tr>
-        <tr>
-            <td>mC14R</td>
-            <td>mRNA for CinR </td>
-        </tr>
-        <tr>
-            <td>C14R</td>
-            <td>CinR transcriptional regulator</td>
-        </tr>
-        <tr>
-            <td>C14Comp</td>
-            <td>Complex by C14 AHL and CinR transcriptional regulator</td>
-        </tr>
-        <tr>
-            <td>g_ANTISTARC14Comp</td>
-            <td>Complex formed between plasmid backbone for ANTISTAR (pCin) and C14 Comp</td>
-        </tr>
-        <tr>
-            <td>g_STAR</td>
-            <td>Copy number for plasmid backbone housing STAR</td>
-        </tr>
-        <tr>
-            <td>STAR</td>
-            <td>Short transcription activating RNA (STAR)</td>
-        </tr>
-        <tr>
-            <td>g_ANTISTAR</td>
-            <td>Copy number for plasmid backbone housing ANTISTAR</td>
-        </tr>
-        <tr>
-            <td>ANTISTAR</td>
-            <td>Anti Short transcription activating RNA (ANTISTAR) </td>
-        </tr>
-        <tr>
-            <td>STAR:ANTISTAR</td>
-            <td>STAR:ANTISTAR Complex</td>
-        </tr>
-        <tr>
-            <td>STAR_Target</td>
-            <td>Star Target plasmid (AD1 terminator)</td>
-        </tr>
-</tbody>
-</table>
-<specialh4> Table 2: Unique Species for GP0.4 model Table </specialh4>
+<div class="column sixth_size">
-<table class="table table-bordered table-hover">
+<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>
-<thead>
+</div>
-        <tr>
-            <th>Species Name</th>
-            <th>Description</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>g_GP0.4</td>
-            <td>Plasmid hosting STAR Target and growth regulating protein GP2</td>
-        </tr>
-        <tr>
-            <td>mGP0.4</td>
-            <td>mRNA for GP0.4</td>
-        </tr>
-        <tr>
-            <td>pre_GP0.4</td>
-            <td>Unfolded GP0.4</td>
-        </tr>
-        <tr>
-            <td>GP0.4</td>
-            <td>GP0.4 (Gene Product 0.4) sequesters FtsZ and stops cell division.</td>
-        </tr>
-        <tr>
-            <td>FtsZ</td>
-            <td>FtsZ is a protein that influences cell division</td>
-        </tr>
-        <tr>
-            <td>GP0.4:FtsZ</td>
-            <td>GP0.4:FtsZ Complex</td>
-        </tr>
+<div class="column sixth_size">
+<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>
+</div>
-</tbody>
-</table>
+<br>
-<specialh4> Table 3: Unique Species for GP2 model Table </specialh4>
-<table class="table table-bordered table-hover">
-<thead>
-        <tr>
-            <th>Species Name</th>
-            <th>Description</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>g_GP2</td>
-            <td>Plasmid hosting STAR Target and growth regulating protein GP2</td>
-        </tr>
-        <tr>
-            <td>mGP2</td>
-            <td>mRNA for GP2</td>
-        </tr>
-        <tr>
-            <td>pre_GP2</td>
-            <td>Unfolded GP2</td>
-        </tr>
-        <tr>
-            <td>GP2</td>
-            <td>GP2 (Gene Product 2) sequesters RNA Polymerase </td>
-        </tr>
-        <tr>
-            <td>RNAP</td>
-            <td>RNA Polymerase</td>
-        </tr>
-        <tr>
-            <td>GP2:RNAP</td>
-            <td>GP2:RNAP Complex</td>
-        </tr>
-</tbody>
+<div class="clear"></div>
-</table>
-<specialh4> Table 4: Unique Species for CAT and LeuB </specialh4>
-<table class="table table-bordered table-hover">
-<thead>
-        <tr>
-            <th>Species Name</th>
-            <th>Description</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>mTet</td>
-            <td>mRNA for Tetracycline</td>
-        </tr>
-        <tr>
-            <td>Tet</td>
-            <td>Tetracycline </td>
-        </tr>
-        <tr>
-            <td>pTet</td>
-            <td>Promoter that is repressed by Tetracycline</td>
-        </tr>
-        <tr>
-            <td>Tet:pTet</td>
-            <td>Tetracycline:pTet complex </td>
-        </tr>
-        <tr>
-            <td>mLeuB</td>
-            <td>mRNA for Leucine B</td>
-        </tr>
-        <tr>
-            <td>LeuB</td>
-            <td>Leucine B</td>
-        </tr>
-        <tr>
-            <td>mCAT</td>
-            <td>mRNA for Chloramphenicol Acetyltransferase</td>
-        </tr>
-        <tr>
-            <td>CAT</td>
-            <td>Chloramphenicol Acetyltransferase</td>
-        </tr>
-</tbody>
-</table>
-     </div>
+<br>
-   </div>
+<div class="naviSection" id="section1">
+<br>
+<br>
+<br>
+<br>
+<br>
+<h2 style="font-family: verdana; font-size: 30px; text-align: center">Part plasmid assembly</h2>
+<br>
+<p style="font-family: verdana">The first part of our Golden Gate assembly workflow was part assembly, in which the <i>gadB</i> gene and the P8/P32 promoters were individually cloned into the entry vector pYTK001 <b>(Fig. 1)</b>. The <i>gadB</i> gene and P8/P32 promoter sequences contain flanking BsmBI sites that produce overhangs compatible with those cut by BsmBI in the entry vector pYTK001. Thus, BsmBI cloning should result in part plasmids containing the <i>gadB</i> gene and P8/P32 promoters set within the pYTK001 backbone. </p>
+<br>
+</html>
+[[File:T--Austin_UTexas--gadBAndPromoterPartPlasmids.jpg|thumb|center|800px|<b>Figure 1.</b> <i>gadB</i> gene and P8/P32 promoter part assembly process. Golden Gate compatible <i>gadB</i> and P8/P32 promoter sequences are cloned into the pYTK001 entry vector via BsmBI assembly.]]
+<html>
+<br>
+<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>
+<br>
+</html>
+[[File:T--Austin_UTexas--PartPlasmidTransformations.jpg|thumb|center|700px|<b>Figure 2.</b> <i>gadB gene</i> and P8/P32 promoter part plasmid <i>E. coli</i> transformations, compared to control transformations with pYTK001. Under UV illumination, transformants containing the correctly assembled part plasmids were non-fluorescent while negative transformants appeared fluorescent like colonies on the control plates.]]
+<html>
+<br>
+<a href="#navtop"><p>Back to Top</p></a>
 </div>
-<div class="some-padding"></div>
+<div class="naviSection" id="section2">
-<div class="some-padding"></div>
+<br>
+<br>
+<br>
+<h2 style="font-family: verdana; font-size: 30px; text-align: center">Testing constitutive <i>Lactococcal</i> promoters in <i>E. coli</i> </h2>
+<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>
+<br>
+<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>
+<br>
+</html>
+[[File:T--Austin_UTexas--p8p32test.jpg|thumb|center|800px|<b>Figure 3.</b> Golden Gate assembly process of the P8 and P32 test cassette plasmids.]]
+<html>
+<br>
+<p style="font-family: verdana">Upon first look, <i>E. coli</i> colonies transformed with these assemblies appeared purple-blue in color. This phenotype was due to the expression of the red fluorescent protein. Further, we noticed that <i>E. coli</i> colonies transformed with these assemblies fluoresced red under UV light, indicating that <b>the P8 and P32 promoters are indeed expressing the <i>E2-Crimson</i> reporter gene and thus are functional in <i>E. coli</i> <b>(Fig. 4).</b> </b></p>
+<br>
+<br>
+</html>
+[[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.]]
+<html>
+<br>
-<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
+<a href="#navtop"><p>Back to Top</p></a>
-             <div class="panel panel-default">
-                <div class="panel-heading" role="tab" id="ParaAcc">
-                    <h4 class="panel-title">
-                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#ParaAcc-collapse" aria-expanded="false" aria-controls="ParaAcc-collapse">
-<div>
-                    <div class="col-md-11">Parameters</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 </div>
-                    </a>
-                    </h4>
-                </div>
+<div class="naviSection" id="section3">
-                <div id="ParaAcc-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="ParaAcc">
-                    <div class="panel-body">
-<specialh4> Table 5: General Numbers Table </specialh4>
+<br>
+<br>
+<br>
-<table class="table table-bordered table-hover">
+<h2 style="font-family: verdana; font-size: 30px; text-align: center">Testing <i>gadB</i> overexpression in <i>E. coli</i></h2>
-<thead>
+<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>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>Cell Volume</td>
-            <td></td>
-            <td>6.70E<sup>-16</sup></td>
-            <td>L</td>
-            <td>Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Vol 1. pp. 15, ASM Press 1996.</td>
-        </tr>
-        <tr>
-            <td>Transcription rate</td>
-            <td>28 Nucleotides/s was used</td>
-            <td>28-89</td>
-            <td>Nucleotides/s</td>
-            <td>Vogel U, Jensen KF. The RNA chain elongation rate in Escherichia coli depends on the growth rate. J Bacteriol. 1994 May176(10):2807-13 p.2811 table 2</td>
-        </tr>
-        <tr>
-            <td>Translation rate</td>
-            <td>12 Amino acid/s was used</td>
-            <td>12-21</td>
-            <td>Amino acids/s</td>
-            <td>Bremer, H., Dennis, P. P. (1996) Modulation of chemical composition and other parameters of the cell by growth rate. Neidhardt, et al. eds. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd ed. chapter 97, p. 1559, Table 3</td>
-        </tr>
-        <tr>
-            <td>d_prot</td>
-            <td>Protein degradation</td>
-            <td>1.39E<sup>-05</sup></td>
-            <td>1/min</td>
-            <td>Biomolecular Systems by Del Vecchio</td>
-        </tr>
-        <tr>
-            <td>k_mat</td>
-            <td>Protein maturation rate</td>
-            <td>0.16</td>
-            <td>1/min</td>
-            <td>Megerle JA, Fritz G, Gerland U, Jung K, Rädler JO. Timing and dynamics of single cell gene expression in the arabinose utilization system. Biophys J. 2008 Aug95(4):2103-15. p.2106 right column bottom of paragraph</td>
-        </tr>
-        <tr>
-            <td>D</td>
-            <td>Dilution rate</td>
-            <td>0.048</td>
-            <td>1/min</td>
-            <td>A synthetic Escherichia coli predator–prey ecosystem</td>
-        </tr>
-        <tr>
-            <td>d_mRNA</td>
-            <td>mRNA degradation rate constant</td>
-            <td>0.139</td>
-            <td>1/min</td>
-            <td>Taken from 5 min mRNA degradation time</td>
-        </tr>
-        <tr>
-            <td>Copy number for 3K3 and 3C3</td>
-            <td>Copy number</td>
-            <td>20-30</td>
-            <td>molecules</td>
-            <td>http://parts.igem.org/Part:pSB3K3</td>
-        </tr>
-        <tr>
-            <td>Copy number for 1K3 and 1C3</td>
-            <td>Copy number</td>
-            <td>100-300</td>
-            <td>molecules</td>
-            <td>http://parts.igem.org/Part:pSB1C3</td>
-        </tr>
-</tbody>
+<br>
-</table>
+</html>
+[[File:T--Austin_UTexas--gadBTestCassette.jpg|thumb|center|800px|<b>Figure 5.</b> Golden Gate assembly process of P8/<i>gadB</i> and P32/<i>gadB</i> test cassette plasmids.]]
+<html>
-<specialh4> Table 6: Quorum Module Numbers Table </specialh4>
-<table class="table table-bordered table-hover">
+<br>
-<thead>
+<br>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td>k_C4</td>
-            <td>Rhl/C4 Production Rate Constant</td>
-            <td><ul>
-<li>8.00E<sup>-07</sup></li>
-<li>5.36E<sup>-13</sup></li>
-<li>5.36E<sup>-22</sup></li>
-<li>3.22E<sup>02</sup></li>
-</ul></td>
-            <td><ul>
-<li>M/min</li>
-<li>nMol/min</li>
-<li>mol/min</li>
-<li>molecules/min</li>
-</ul></td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-<tr>
-            <td>k_C14</td>
-            <td>Cin/C14 Production Rate Constant</td>
-            <td><ul>
-<li>5.00E<sup>-08</sup></li>
-<li>3.35E<sup>-14</sup></li>
-<li>3.35E<sup>-23</sup></li>
-<li>2.00E<sup>01</sup></li>
-</ul></td>
-            <td><ul>
-<li>M/min</li>
-<li>nMol/min</li>
-<li>mol/min</li>
-<li>molecules/min</li>
-</ul></td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-<tr>
+<p style="font-family: verdana">Similar to the pYTK001 entry vector in part assembly, the pYTK095 backbone used for cassette assembly contained a <i>gfp</i> reporter gene that is replaced by sequences of interest. This allowed us to easily perform a phenotypic screen for positive colonies. <b>Non-fluorescent colonies may potentially have had the correct cassette assembly, while fluorescent colonies did not (Fig. 6).</b></p>
-            <td>k_mC4R</td>
-            <td>Transcription rate of C4R</td>
-            <td><ul>
-<li>2.23</li>
-<li>5.51E<sup>-09</sup></li>
-<li>3.69E<sup>-24</sup></li>
-</ul></td>
-            <td><ul>
-<li>mRNA/min</li>
-<li>nMol/min</li>
-<li>Mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-<tr>
-            <td>k_mC14R</td>
-            <td>Transcription rate of C14R</td>
-            <td><ul>
-<li>2.23</li>
-<li>3.69E<sup>-15</sup></li>
-<li>3.69E<sup>-24</sup></li>
-</ul></td>
-            <td><ul>
-<li>mRNA/min</li>
-<li>nMol/min</li>
-<li>Mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-<tr>
+</html>
-            <td>k_C4R</td>
+[[File:Greenwhitescreen.png|thumb|center|800px|<b>Figure 6.</b> P8/<i>gadB</i> and P32/<i>gadB</i> cassette E. coli transformations alongside a pYTK095 control transformation under UV illumination. Potentially positive colonies containing the correct assemblies appeared non-fluorescent, while negative colonies appeared fluorescent.
-            <td>Translation rate of C4R</td>
+]]
-            <td><ul>
+<html>
-<li>2.99</li>
-<li>4.96E<sup>-24</sup></li>
-</ul></td>
-            <td><ul>
-<li>Protein/min</li>
-<li>Mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average translation rate : Bremer et al</td>
-        </tr>
-<tr>
-            <td>k_C14R</td>
-            <td>Translation rate of C14R</td>
-            <td><ul>
-<li>2.99</li>
-<li>4.96E<sup>-24</sup></li>
-</ul></td>
-            <td><ul>
-<li>Protein/min</li>
-<li>Mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average translation rate : Bremer et al</td>
-        </tr>
-<tr>
+<p style="font-family: verdana">The non-fluorescent colonies were then screened using colony PCR, and positive colonies were then miniprepped and sequenced. <b>The sequencing results indicated that there were several mutations within the <i>gadB</i> gene in the samples.</b> These mutations are recorded in <b>Table 1.</b></p>
-            <td>kf_C4Comp</td>
-            <td>C4 - C4R Complex formation</td>
-            <td><ul>
-<li>1.00E<sup>08</sup></li>
-<li>2.50E<sup>-01</sup></li>
-</ul></td>
-            <td><ul>
-<li>1/M.min</li>
-<li>1/complexes.min</li>
-</ul></td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
-            <td>kr_C4Comp</td>
-            <td>C4:C4R Complex dissassociation</td>
-            <td>10</td>
-            <td>1/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-<tr>
+</html>
-            <td>kf_C14Comp</td>
+[[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. ]]
-            <td>C4 - C4R Complex formation</td>
+<html>
-            <td><ul>
-<li>1.00E<sup>08>/sup></li>
-<li>2.50E<sup>-01</sup></li>
-</ul></td>
-            <td><ul>
-<li>1/M.min</li>
-<li>1/complexes.min</li>
-</ul></td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
+<br>
-            <td>kr_C14Comp</td>
-            <td>C4:C4R Complex dissassociation</td>
-            <td>10</td>
-            <td>1/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
-            <td>kf_DimC4</td>
-            <td>C4:C4R complex dimerization</td>
-            <td>5.00E<sup>07</sup></td>
-            <td>M/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
+<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>
-            <td>kr_DimC4</td>
-            <td>C4:C4R complex reverse dimerization</td>
-            <td>1</td>
-            <td>1/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
+<br>
-            <td>kf_DimC14</td>
-            <td>C14:C14R complex dimerization</td>
-            <td>5.00E<sup>07</sup></td>
-            <td>M/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
+<p style="font-family: verdana"><b>To troubleshoot this problem, we decided to use a backbone containing the low-copy number p15A origin for the cassette assembly (Fig. 7).</b></p>
-            <td>kr_DimC14</td>
-            <td>C14:C14R complex reverse dimerization</td>
-            <td>1</td>
-            <td>1/min</td>
-            <td>Dynamics of quorum sensing switch - Weber</td>
-        </tr>
-        <tr>
-            <td>d_C4</td>
-            <td>C4 degradation rate constant</td>
-            <td>2.21E<sup>-04</sup></td>
-            <td>1/min</td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-        <tr>
-            <td>d_C14</td>
-            <td>C14 degradation rate constant</td>
-            <td>2.83E<sup>-04</sup></td>
-            <td>1/min</td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-        <tr>
+</html>
-            <td>d_C4R</td>
+[[File:T--Austin_UTexas--TestCassetteLowCopy.jpg|thumb|center|700px|<b>Figure 7.</b> Golden Gate assembly process of P8/<i>gadB</i> and P32/<i>gadB</i> test cassette plasmids using a backbone containing the low-copy number p15A origin.]]
-            <td>C4R degradation rate constant</td>
+<html>
-            <td>0.002</td>
-            <td>1/min</td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-        <tr>
-            <td>d_C14R</td>
-            <td>C14R degradation rate constant</td>
-            <td>0.002</td>
-            <td>1/min</td>
-            <td>Optimal tuning of bacterial sensing potential - Anand Pai & Lingchong You</td>
-        </tr>
-</tbody>
-</table>
-<specialh4> Table 7: STAR Module Numbers Table </specialh4>
+<p style="font-family: verdana">Copy number reduction of our <i>gadB</i> overexpression cassette plasmids (accomplished by replacing the ColE1 origin with the p15A origin) was expected to decrease the overall amount of <i>gadB</i> expression, thereby decreasing the metabolic load imposed on the cells and the mutation rates observed in the <i>gadB</i> gene. <b>However, we also observed several mutations within the <i>gadB</i> gene in the potentially positive cassette plasmids sequenced.</b> The observed mutations are detailed in Table 2.</p>
-<table class="table table-bordered table-hover">
+</html>
-<thead>
+[[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.]]
-        <tr>
+<html>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+<br>
-            <td>kf_gSTARC4Comp</td>
-            <td>gSTAR-C4 comple association</td>
-            <td>0.25</td>
-            <td>1/molecules.min</td>
-            <td>Assumed same as quorum complex formation</td>
-        </tr>
-        <tr>
+<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>
-            <td>kr_gSTARC4Comp</td>
-            <td>gSTAR-C4 complex disassociation</td>
-            <td>10</td>
-            <td>1/min</td>
-            <td>Assumed same as quorum complex formation</td>
-        </tr>
-        <tr>
-            <td>kf_gANTISTARC14Comp</td>
-            <td>gANTISTAR-C14 complex association</td>
-            <td>0.25</td>
-            <td>1/molecules.min</td>
-            <td>Assumed same as quorum complex formation</td>
-        </tr>
-        <tr>
+</html>
-            <td>kr_gANTISTARC14Comp</td>
+[[File:T--Austin_UTexas--iptginduciblegadB.jpg|thumb|center|800px|<b>Figure 8.</b> Golden Gate assembly process of IPTG-inducible <i>gadB</i> expression cassette plasmid.]]
-            <td>gANTISTAR - C14 complex disassociation</td>
+<html>
-            <td>10</td>
-            <td>1/min</td>
-            <td>Assumed same as quorum complex formation</td>
-        </tr>
-        <tr>
+<a href="#navtop"><p>Back to Top</p></a>
-            <td>k_STAR</td>
+</div>
-            <td>Basal Rate of STAR production (without C4:C4R induction)</td>
+<br>
-            <td><ul>
-<li>1.91E<sup>-01</sup></li>
-<li>3.08E<sup>-23</sup></li>
-</ul></td>
-            <td><ul>
-<li>mRNA/min</li>
-<li>mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
+<div class="naviSection" id="section4">
+<br>
+<br>
+<br>
-        <tr>
+<h2 style="font-family: verdana; font-size: 28px; text-align: center">Creating a Golden Gate compatible shuttle vector</h2>
-            <td>k_iSTAR</td>
-            <td>Rate of STAR production (after induction of C4:C4R)</td>
-            <td><ul>
-<li>18.57</li>
-<li>3.08E<sup>-23</sup></li>
-</ul></td>
-            <td><ul>
-<li>mRNA/min</li>
-<li>mol/min</li>
-</ul></td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<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>
-            <td>k_ANTISTAR</td>
-            <td>Basal Rate of ANTISTAR production (without C14:C14R induction)</td>
-            <td>1.91E<sup>-01</sup></td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<br>
-            <td>k_iANTISTAR</td>
-            <td>Rate of ANTI STAR production (after induction of C14:C14R)</td>
-            <td>18.57</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+</html>
-            <td>kf_STARANTISTAR</td>
+[[File:T--Austin_UTexas--Final_Cassette.jpg|thumb|center|800px|<b>Figure 9.</b> Golden Gate assembly of the GABA final overexpression cassette plasmid with the Golden Gate compatible pMSP3535 vector and the P8/P32 promoter, <i>gadB</i> gene, and M13 terminator part plasmids.]]
-            <td>STAR:ANTISTAR complex formation</td>
+<html>
-            <td>62</td>
-            <td>complexes/min</td>
-            <td>Calculated using DNA strand displacement (Winfree et al)</td>
-        </tr>
-        <tr>
+<br>
-            <td>kr_STARANTISTAR</td>
-            <td>STAR:ANTISTAR complex dissociation</td>
-            <td>2</td>
-            <td>1/min</td>
-            <td>Calculated using DNA strand displacement (Winfree et al)</td>
-        </tr>
-        <tr>
+<p style="font-family: verdana">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 <b>(Fig. 10).</b></p>
-            <td>kf_STARAD1</td>
-            <td>STAR:AD1 Complex formation</td>
-            <td>62</td>
-            <td>complexes/min</td>
-            <td>Calculated using DNA strand displacement (Winfree et al)</td>
-        </tr>
-        <tr>
+<br>
-            <td>kr_STARAD1</td>
-            <td>STAR:AD1 Complex dissociation</td>
-            <td>2</td>
-            <td>1/min</td>
-            <td>Calculated using DNA strand displacement (Winfree et al)</td>
-        </tr>
-</tbody>
+</html>
-</table>
+[[File:T--Austin_UTexas--CreatingpMSP3535GGA.jpg|thumb|center|800px|<b>Figure 10.</b> Assembly workflow for creating the Golden Gate compatible pMSP3535 backbone. The first step involves replacing the ColE1 origin in the pMSP3535 backbone with a new ColE1 origin from pYTK001 to create a pMSP3535 + ColE1 assembly. The second step involves combining the pMSP3535 + ColE1 assembly with a <i>gfp</i> dropout to form the final Golden Gate compatible vector to be used to create our GABA overexpression plasmid.]]
+<html>
+<br>
-<specialh4> Table 8: Gp0.4 Module Numbers Table </specialh4>
+<p style="font-family: verdana"><b>Since the original pMSP3535 vector contained two illegal BsaI sites within the ColE1 origin, we sought to replace this ColE1 origin with a BsaI-free one isolated from pYTK001.</b> This assembly process involved linearizing and adding BsmBI sites and compatible overhangs to the pMSP3535 backbone and the pYTK001 ColE1 origin via PCR. After the pMSP3535 backbone and ColE1 origin were successfully amplified by PCR <b>(Fig. 11a)</b>, they were joined together using BsmBI assembly. Diagnostic PCR was performed on pMSP3535 + ColE1 minipreps from <i>E. coli</i> transformants to screen for positive samples containing both the pMSP3535 backbone and the ColE1 inserts (Fig. 11b). <b>After confirming the presence of the pMSP3535 vector and ColE1 origin, we partially sequence-confirmed the two miniprep samples.</b></p>
-<table class="table table-bordered table-hover">
+<br>
-<thead>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+</html>
-            <td>k_mGP0.4</td>
+[[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.]]
-            <td>Transcription of mGP0.4 before STAR induction</td>
+<html>
-            <td>0.086</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<br>
-            <td>k_imGP0.4</td>
-            <td>Transcription of mGP0.4 after STAR induction</td>
-            <td>8.08</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<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>
-            <td>kf_GP0.4Ftsz</td>
-            <td>GP0.4:Ftsz complex formation</td>
-            <td>9.44E<sup>03</sup></td>
-            <td>1/molecules.min</td>
-            <td></td>
-        </tr>
-        <tr>
+<br>
-            <td>kr_GP0.4Ftsz</td>
-            <td>GP0.4:Ftsz complex dissociation</td>
-            <td>30</td>
-            <td>1/min</td>
-            <td></td>
-        </tr>
-        <tr>
+</html>
-            <td>k_GP0.4</td>
+[[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.]]
-            <td>Translation of GP0.4</td>
+<html>
-            <td>13.86</td>
-            <td>Protein/min</td>
-            <td>Calculated from Bionumbers average translation rate : Bremer et al</td>
-        </tr>
-</tbody>
+<br>
-</table>
-<specialh4> Table 9: Tet Module Numbers Table </specialh4>
+<h4 style="font-family: verdana"; font-size: 28px; text-align: center">Assessing erythromycin susceptibility of <i>E. coli</i></h4>
-<table class="table table-bordered table-hover">
+<br>
-<thead>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+<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>
-            <td>k_mTET</td>
-            <td>Basal transcription of TET</td>
-            <td>0.028</td>
-            <td>mRNA/min</td>
-            <td>Calculated from experimental results for STAR activation (94 fold activation)</td>
-        </tr>
-        <tr>
+<br>
-            <td>k_imTET</td>
-            <td>Induced transcription of TET</td>
-            <td>2.7</td>
-            <td>mRNA/min</td>
-            <td></td>
-        </tr>
-        <tr>
+</html>
-            <td>k_TET:pTET</td>
+[[File:T--Austin_UTexas--erymic.jpg|thumb|center|800px|<b>Figure 13.</b> Erythromycin minimum inhibitory concentration tests for <i>E. coli</i> in liquid media. From left to right: 0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL, 350 µg/mL, and 400 µg/mL.]]
-            <td>TET:pTET complex formation</td>
+<html>
-            <td>120</td>
-            <td>1/molecules.min</td>
-            <td><a href="https://2009.igem.org/Team:Aberdeen_Scotland/parameters/invest_1">https://2009.igem.org/Team:Aberdeen_Scotland/parameters/invest_1</a></td>
-        </tr>
-        <tr>
+<a href="#navtop"><p>Back to Top</p></a>
-            <td>k_-TET:pTET</td>
+</div>
-            <td>TET:pTET complex dissassociation</td>
-            <td>8.40E<sup>05</sup></td>
-            <td>1/min</td>
-            <td><a href="https://2009.igem.org/Team:Aberdeen_Scotland/parameters/invest_1">https://2009.igem.org/Team:Aberdeen_Scotland/parameters/invest_1</a></td>
-        </tr>
-</tbody>
+<br>
-</table>
-<specialh4> Table 10: LeuB Module Numbers Table </specialh4>
+<div class="naviSection" id="section5">
+<br>
+<br>
+<br>
+<div style="background-color: lightcyan;padding:14px;">
+<h2 style="font-family: verdana; font-size: 30px; text-align: center"><i>Lactobacillus plantarum</i> transformation</h2>
-<table class="table table-bordered table-hover">
+<br>
-<thead>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+<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>
-            <td>k_mLeuB</td>
-            <td>Basal transcription of LeuB transcription</td>
-            <td>1.538</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<br>
-            <td>k_imLeuB</td>
-            <td>Induced transcription of LeuB transcription</td>
-            <td>0.015</td>
-            <td>mRNA/min</td>
-            <td>99% Repression from tet was assumed</td>
-        </tr>
+<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 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>
-        <tr>
+<br>
-            <td>k_LeuB</td>
-            <td>Translation of LeuB</td>
-            <td>0.0218</td>
-            <td>molecule/min</td>
-            <td><a href="http://biocyc.org/gene?orgid=ECOLI&id=3-ISOPROPYLMALDEHYDROG-MONOMER#tab=FTRS">http://biocyc.org/gene?orgid=ECOLI&id=3-ISOPROPYLMALDEHYDROG-MONOMER#tab=FTRS</a></td>
-        </tr>
-</tbody>
+</html>
-</table>
+[[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.]]
+<html>
-<specialh4> Table 11: CAT Module Numbers Table </specialh4>
+<br>
-<table class="table table-bordered table-hover">
+<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>
-<thead>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+<strong><p style="font-family: verdana"> Genomic and plasmid sequence verification is underway.</p></strong>
-            <td>k_mCAT</td>
-            <td>Basal transcription of CAT transcription</td>
-            <td>1.875</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
-        <tr>
+<br>
-            <td>k_imCAT</td>
-            <td>Induced transcription of CAT transcription</td>
-            <td>0.018</td>
-            <td>mRNA/min</td>
-            <td>99% Repression from CAT was assumed</td>
-        </tr>
+</html>
+[[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.'']]
+<html>
+<br>
+</html>
+[[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.'']]
+<html>
+<br>
+<h2 style="font-family: verdana; font-size: 30px; text-align: center">References</h2>
+<br>
-        <tr>
+<ol style="font-size:13px; font-family: verdana">
-            <td>k_CAT</td>
+<li>Yunes, R. A et al. GABA production and structure of <i>gadB</i>/<i>gadC</i> genes in <i>Lactobacillus</i> and <i>Bifidobacterium</i> strains from human microbiota. <i>Anaerobe</i>. 42: 197-204 (2016).</li>
-            <td>Translation of CAT</td>
-            <td>0.03568</td>
+<li>Zhu, D. et al. Isolation of strong constitutive promoters from <i>Lactococcus lactis</i> subsp. Lactis N8. <i>FEMS Microbiol Lett</i>. 363(16): pii: fnv107 (2015). </li>
-            <td>molecule/min</td>
-            <td><a href="http://www.uniprot.org/uniprot/P62577">http://www.uniprot.org/uniprot/P62577</a></td>
+<li>Lee, M. E. et al. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. <i>ACS Synth. Biol</i>. 4(9): 975-86 (2015). </li>
-        </tr>
-</tbody>
+<li>Commichau, F. M., et al. Glutamate metabolism in <i>Bacillus subtilis</i>: Gene Expression and Enzyme Activities Evolved To Avoid Futile Cycles and to allow Rapid Responses to Perturbations of the System. <i>AMS</i>. 190(10): 3557-64 (2008).</li>
-</table>
+<li>Sarkar, P.K. and Nout, M.J.R. (Eds.) 2014. Handbook of Indigenous Foods Involving Alkaline Fermentation. CRC Press/Taylor & Francis Group, p. 629.</il>
-<specialh4> Table 12: GP2 Module Numbers Table </specialh4>
+<li>Sleight, S. C. et al. Designing and engineering evolutionary robust genetic circuits. Journal of Biological Engineering. (4):12 (2010).</il>
+<li>Pérez-Arellano, I. et al. Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and <i>Escherichia coli</i>. Plasmid. 46(2): 106-16 (2001).</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>
-<table class="table table-bordered table-hover">
+<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>
-<thead>
-        <tr>
-            <th>Parameter name</th>
-            <th>Description</th>
-            <th>Value</th>
-            <th>Unit</th>
-            <th>Source</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
+<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>
-            <td>k_mGP2</td>
-            <td>Transcription of mGP2 before STAR induction - Basal</td>
-            <td>0.07</td>
-            <td>mRNA/min</td>
-            <td>Calculated using 94 fold activation </td>
-        </tr>
-        <tr>
+<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>
-            <td>k_imGP2</td>
-            <td>Transcription of mGP2 after STAR induction (7.2 kDa)</td>
-            <td>6.56</td>
-            <td>mRNA/min</td>
-            <td>Calculated from Bionumbers average transcription rate - RNA polymerase transcription</td>
-        </tr>
+<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>
-        <tr>
+<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>
-            <td>k_GP2RNAP</td>
-            <td>GP2:RNAP complex formation</td>
-            <td></td>
-            <td>1/Mole.min</td>
-            <td>Intracellular Kinetics of a Growing Virus: A Genetically Structured Simulation for Bacteriophage T7</td>
-        </tr>
-        <tr>
-            <td>k_-GP2RNAP</td>
-            <td>GP2:RNAP complex dissociation</td>
-            <td></td>
-            <td>1/min</td>
-            <td>Intracellular Kinetics of a Growing Virus: A Genetically Structured Simulation for Bacteriophage T7</td>
-        </tr>
-        <tr>
+<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>
-            <td>k_GP2</td>
-            <td>Translation of GP2</td>
-            <td>11.2</td>
-            <td>Protein/min</td>
-            <td>Intracellular Kinetics of a Growing Virus: A Genetically Structured Simulation for Bacteriophage T7</td>
-        </tr>
-        <tr>
-            <td>RNA</td>
-            <td>Total RNA pol</td>
-            <td>1800</td>
-            <td>molecules/cell</td>
-            <td>Intracellular Kinetics of a Growing Virus: A Genetically Structured Simulation for Bacteriophage T7</td>
-        </tr>
-        <tr>
+<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>
-            <td>k_mRNAP</td>
-            <td>Transcription of RNA pol</td>
-            <td>2400</td>
-            <td>nucleotides/min.RNAP</td>
-            <td>Intracellular Kinetics of a Growing Virus: A Genetically Structured Simulation for Bacteriophage T7</td>
-        </tr>
-</tbody>
-</table>
-     </div>
+</ol>
-   </div>
+<a href="#navtop"><p>Back to Top</p></a>
 </div>
-</section>
-</body>
-<img class="backTop" onclick="document.documentElement.scrollTop = document.body.scrollTop = 500;" src="https://static.igem.org/mediawiki/2016/1/19/T--Imperial_College--BackTop.png" >
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