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| | Figure 10 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states. Thus, the toggle switch is able to choose a random state and no state is significantly more preferred than the other . This is in contrast to what the model predicted. | | Figure 10 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states. Thus, the toggle switch is able to choose a random state and no state is significantly more preferred than the other . This is in contrast to what the model predicted. |
| | </p> | | </p> |
| - | <br/>
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| - | <h2 id="promoterdesign">Promoter Design</h2>
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| - | <p>
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| - | The model of the system we build predicted that the system would not be stable with the promoter configurations we had been using so far. These promoters, see figure 11, where promoters from the registry with the same operator sites that we needed, but configured in such a way that it was likely they would not work. This was predicted by the moddel of the kilswitch . Therefore promoters with another configuration of the operator sites of the promoter would be necessary. With the results of the model we build 6 new promoters based on the sequences of Elowiz et al 2007 [2].
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| - | </p>
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| - | <figure>
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| - | <img src="https://static.igem.org/mediawiki/2014/e/e6/Wageningen_UR_killswitch_Pic11.png" width="80%">
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| - | <figcaption> Figure 11: the overview of the kill switch regulatory system. The promoters are circled .
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| - | </figcaption>
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| - | </figure><br/>
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| - | <p>
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| - | Promoters have 3 different regions that are separated by the -34 and -10 RNA polymerase binding sites, see figure 12. In the article Elowiz et al 2007 [2] these are called the distal, core and proximal region.
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| - | </p>
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| - | <figure>
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| - | <img src="https://static.igem.org/mediawiki/2014/3/32/Wageningen_UR_killswitch_Pic12.png" width="80%">
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| - | <figcaption> Figure 12: a schematic representation of promoter design. The promoter region is divided into three sections: the distal, core and proximal region separated by the -34 and -10 RNA polymerase binding sites. The distal, core and proximal regions can contain different operator sites such as the tetR (orange), LacI (yellow) and CI (blue) operator site or no operator site (empty site). We made 6 different combinations of these operator sites providing two CI/Lac promoters, two CI/Tet promoters and tow Tet promoters.
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| - | </figcaption>
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| - | </figure><br/>
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| - | <p>
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| - | Binding sites on the core, distal and proximal regions have different strengths, generally the core region is the strongest followed by the proximal region and finally the distal region [2] [3]. By putting repressor operator sites on different positions you can influence the strength of the repression [2, 3] and place the most important repressor sites on the strongest regions. In theory systems would be tuneable with this method. Here we give this theory a try by designing promoters for a toggle switch that have operator sites at the same places so that the toggle switch is as balanced as possible.the most stable setup was modelled and the results can be found here . The promoters from the registry where not balanced in this way, so we made new promoters with the sequences provided in the article of Elowiz et al 2007 [2]. In the article a set of different sequences for the proximal, core and distal regions was used that can be combined in different combinations. We used these sequences to design 6 different promoters <i>in silico/<i>. The operator configurations of the 6 promoters are listed below:
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| - | <ul>
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| - | <li>Empty site, TetR site, TetR site (-- Tet Tet)</li>
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| - | <li>TetR site, empty site, TetR site (Tet – Tet)</li>
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| - | <li>CIλ site, LacI site, LacI site (CI Lac Lac)</li>
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| - | <li>TetR site, CIλ site, TetR site (Tet CI Tet)</li>
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| - | <li>CIλ site, TetR site, TetR site (CI Tet Tet)</li>
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| - | <li>LacI site, CIλ site, Lac site (Lac CI Lac)</li>
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| - | </ul>
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| - | </p>
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| - | <p>
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| - | We then ordered two single stranded sequences that had an overlapping region, and used these to make a double stranded promoter with prefix and suffix so they can be assembled according to BioBrick Standard 10. These promoters where ten assembled into pSB1C3 and other plasmids to make the kill-switch system. The protocol can be found here.
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| - | </p>
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| - | <br/>
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| - | <h2 id="results3">Results</h2>
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| - | <p>
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| - | We were able to assemble the promoters with GFP, to characterize their functionality after rhamnose induction. All promoters except for promoter 3 (CI Lac Lac) showed GFP expression. See also the plates depicted in Figure 13.
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| - | </p>
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| - | <p>
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| - | Figure 13 shows all the promoters (P1-6) with GFP on ampicillin plates exposed to UV light. On each plate are colonies that are fluorescent suggesting correct constructs in these bacteria, except for promoter 3. After sequencing all parts had the expected sequences so promoter 3 is either a very weak promoter or the sequence used is not working. Future work with a plate reader could give some more conclusive results.
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| - | </p>
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| - | <br/>
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| - | <h2 id="characterization">Rhamnose Mediated Characterization</h2>
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| - | <p>
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| - | Some well-known promoters like the Lac promoter and the Tet promoter have a repressor that can be inactivated by a chemical, making characterization relatively easy. In this way the promoter can be induced by this chemical. By treatment of different concentrations of this chemical there will be different expression levels of this promoter. With this information you can compare different promoters. The drawback of this is that you need a promoter that is inducible with a chemical. There are also repressors and promoters where that is not possible such as the CIλ promoter and repressor. The CI lambda repressor is not inhibited by a chemical so the CI lambda promoter is not inducible. To characterize non inducible promoters we developed a new system to characterize promoters based on a two plasmid system with a ramose promoter and GFP, see Figure 14.
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| - | </p>
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| - | <p>
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| - | The final design to characterize promoters includes the following two components:
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| - | <ul>
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| - | <li>A repressor plasmid with a rhamnose promoter and a repressor of choice</li>
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| - | <li>A promoter GFP plasmid with your favoured promoter and GFP</li>
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| - | </ul>
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| - | </p>
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| - | <p>
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| - | The double repressible promoters used in the kill switch system are taken from the iGEM Registry. Since these promoters pCI/lac and pCI/tet were not characterized we could not estimate if a functional kill switch could be constructed with these promoters.
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| - | The double repressible promoter pCI/lac from the toggle switch, is characterized. pCI/lac was combined with a <i>gfp</i> reporter gene under Elowitz RBS (Bba_I13504 ) to make the new BioBrick pCI/lac <i>gfp</i> . As a reference pRha and the tetracyclin promoter pTet are also assembled to the <i>gfp</i> gene (BBa_K1493501, BBa_K1493504 ). Since the promoter strength of pTet is known in relative promoter unit (RPU) it can be used as a reference promoter to estimate promoter strength [4]. In this way, we can compare the constitutive promoter strength at one time point and give the characterized promoter a RPU value. The rhamnose promoter was successfully assembled to the <i>CI</i> (BBa_P0451) and <i>lac</i> (BBa_P0412) repressor protein genes under Elowitz RBS creating new BioBricks BBa_K1493510 and BBa_K1493520.
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| - | </p>
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| - | <br/>
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| - | <h2 id="results4">Results</h2>
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| - | <p>
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| - | The graphs containing all data points can be found in here . The characterization protocol can be found here.
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| - | </p>
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| - | <p>
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| - | The measurements in figure 15 indicate an activation of pRha by L-rhamnose. The RFU values of 0% and 0.001% rhamnose are not significant taking into account the high standard deviation for these measurements as can be seen in figure 15 B. From a rhamnose concentration of 0.01% to 0.2% a significant increase in fluorescence is measured. Fluorescence from figure 15 can be used to predict the concentration of repressor protein produced. Data points for time 8.15 were chosen for the graph in figure 15 B due to the peak at time for 0.2% rhamnose, which is visible in figure 15 A. This peak can be explained by the rhamnose depletion, as it is consumed by <i>E. coli</i>, causing the stop of promoter induction. We assume that at this time point the highest concentration of repressor proteins are present in the cells inhibiting the expression of GFP.
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| - | </p>
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| - | <p>
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| - | The constitutive promoter strength of pCI/lac , is determined using the results of the fluorescence measurements. The RFU values of NEB5α <i>E. coli</i> strains containing pCI/lac GFP and pTet GFP are shown in figure 16. As can be seen in this figure pTet is a stronger promoter than pCI/lac. Using the results shown in figure 16 the RPU value of pCI/Lac is determined using the known value of pTet as stated by Kelly et al. 2009 [4]. Data from time point 8.13 was chosen for calculation. Compared to the pTet RPU value 1.5, pCI/lac has a RPU of 1.0.
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| - | </p>
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| - | <p>
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| - | Figure 17 shows that a higher concentration of L-rhamnose gives a lower RFU value. This means that the GFP expression is repressed by the repressor protein produced under the rhamnose promoter. As shown in figure 15 a low concentration of rhamnose (0.001%) does not have any substantial effect on the expression rate of the protein. This is also visible in figure 17 in which for both CIλ (A) and LacI (B) 0.001% rhamnose doesn’t show a lower fluorescence compared to 0% rhamnose. We expected the same RFU values for cultures grown in medium with 0.2% glucose and 0% rhamnose, since glucose functions as a repressor for pRha. Though, cultures grown in medium with glucose show lower RFU values in both graphs in figure 17. This can be explained by the higher growth rate of the cells in glucose containing medium, since the RFU value is OD dependant (notebook). In figure 15 0.01% rhamnose shows a higher RFU compared to the culture grown on 0% rhamnose, which indicates that pRha is active at that concentration at that time point. However, at the concentration of 0.01% rhamnose both CIλ (A) and LacI (B) don’t show any substantial repression. We can conclude that the concentration repressors produced at the promoter strength of pRha in 0.01% rhamnose is not high enough to perform repression of the promoter pCI/lac. Though, cultures grown in 0.05% and 0.2% rhamnose show a lower RFU value for both constructs as the RFU in figure 15 increases in higher rhamnose concentrations. We can conclude that repression of pCI/lac is stronger in higher concentrations rhamnose, since higher concentrations rhamnose lead to a higher concentration of repressor proteins. We expected the same RFU values for cultures grown in medium with 0.2% glucose.
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| - | </p>
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| - | <br/>
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| - | <h2 id="futurework">Future work</h2>
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| - | <p>
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| - | Future work:
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| - | In the last month we made some characterization plasmids to characterize the registry promoters and the new set of promoters, but due to a lack of time we were not able to finish them all or to characterize them all. In future work they could be finished and the new and registry promoters could be fully characterized. More experiences with this new type of characterization could also further optimize the protocol and the rhamnose concentrations that should be used. When fully developed this could become a standard method to characterize repressible promoters, since it is able to characterize non chemically inducible promoters with different levels of rhamnose.
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| - | The final regulatory system was only a few steps away from completion, but is due to a lack of time not finished. In future work this system could be finished, tested and characterized. When fully finished this system could become a mayor basis for safety systems and regulated expression. It can be implemented as a kill-switch as would be the case for BananaGuard or as regulator for production in a bioreactor where the bacteria only produce products when the rhamnose is cleared out of the media.
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| - | </p>
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| - | <br/>
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| - | <h2 id="partssummary">Parts summary</h2>
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| - | <br/>
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| - | <p>
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| - | <b>New promoter parts</b>
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| - | </p>
| |
| - | <figure>
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| - | <figcaption>
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| - | Table 1: promoter biobricks. The first column contains the biobrick codes the second column explains the function of the biobrick and the third column schematically shows the order of the repressor operator sites within the promoter. The most right column shows a short codename for these promoters.
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| - | </figcaption>
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| - | </figure>
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| - |
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| - |
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| - | <p>
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| - | <b>The promoters from the registry</b>
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| - | </p>
| |
| - | <figure>
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| - | <figcaption>
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| - | Table 2: Promoters used from the registry to build the kill-switch system that later were replaced with new promoters in the system.
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| - | </figcaption>
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| - | </figure>
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| - |
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| - |
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| | <p> | | <p> |
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