Team:IIT Madras/Experiments

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Latest revision as of 00:18, 22 October 2009

Indian Institute of Technology,Madras

Experiments

Comparing the differences in the growth rates of cells with and without plasmids in various media

We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 7.

Fig 7: This experiment helps in comparing the growth rates of various strains in different antibiotic media.

We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.

Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:

DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl
RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl
CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl
RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl

Thus the starting point for all the samples is the same - an OD600 of 0.01. Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.

Experimental protocol:

1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.

2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.

3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).

4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.

5. The whole procedure is repeated to check for reproducibility.

Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.

Click here for the results

Modeling

Basic Equations:

Where,

X⁺ is the cells with the plasmid.

X^- is the cells without the plasmid.

u⁺ is the specific cell growth rate of cells with the plasmid.

u^- is the specific cell growth rate of cells without the plasmid.

p is the probability of a cell to lose a plasmid.

We are trying to come up with a mathematical model for the relative populations of plasmid containing and plasmid free cells with respect to time using the growth curves that have been generated and the results from the fluorescence.

Fluorescence Imaging of cells to check for directed plasmid loss

This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how

Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium
Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.
Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.
Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.

Fig 6.3: Experimental procedure for measuring the rate of plasmid loss by fluorescent imaging

We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3.

RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol
CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.
Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol.

The expected behavior of the system is (listed in the same order as the above 3 conditions):

RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.
CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.
Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.

Protocol:

1. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.
2. Culture is grown for 4 hours or till the OD600 crosses 0.1.
3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).

Required inoculations are: a. Broth 1 into tube with no antibiotic in the LB - tube 1
b. Broth 1 into tube with Ampicillin in the LB - tube 2
c. Broth 2 into tube with no antibioti in the LB - tube 3
d. Broth 2 into tube with Chloramphenicol in the LB - tube 4
e. Broth 3 into tube no antibiotic in the LB - tube 5
f. Broth 3 into tube with Ampicillin in the LB - tube 6
g. Broth 3 into tube with Chloramphenicol in the LB - tube 7
h. Broth 3 into tube with both the antibiotics in the LB - tube 8

4. This culture is allowed to grow for 2.5 hours.
5. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.
6. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes. 7. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.

Click here to see the results of the fluorescence imaging.

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-<p CLASS="justifyalign">Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further.
-<p CLASS="justifyalign">If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).</p>
-</body>
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+==Experiments==
+===Comparing the differences in the growth rates of cells with and without plasmids in various media===
-==Summary==
+<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 7.
-<p CLASS="justifyalign">Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.</p>
-<p CLASS="justifyalign">We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.
 </p>
-<html><br></html>
+[[Image:growth curves.jpg|700px]]
+<html><br>
+<b><font color="#000">Fig 7: </font></b><i>This experiment helps in comparing the growth rates of various strains in different antibiotic media.</i>
+</html>
-[[Image:plasmid network.jpg|650px]]
+<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p>
+<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:
-<html><br></html>
-<p CLASS="justifyalign">
-<b><font color="#000">Fig 1:</font></b><i>The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.</i>
 </p>
+* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl
+* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl
+* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl
+* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl
-<html><br></html><html><br></html>
+<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.
+Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p>
-<p CLASS="justifyalign">
+<b><font color="#000">Experimental protocol:</font></b>
-However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.</p>
-==Theory==
+<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p>
+<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p>
+<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p>
+<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p>
+<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p>
-<p CLASS="justifyalign">The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless it confers a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).</p>
+<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p>
-<html><br></html>
+Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results
-<html><br></html>
-[[Image:segregationandplasmidloss.jpg|300px]]
+===Modeling===
-<html><br></html>
+<html><br>
+<b><font color="#000">Basic Equations:</font></b>
+<br>
+<img src="https://static.igem.org/mediawiki/2009/8/89/Modeling.jpg" align="center" width="400" height="200"></a>
+<br>
-<p CLASS="justifyalign">
+<p>Where,</p>
-<b><font color="#000">Fig 2.1:</font></b> <i>During cell division, rarely all the plasmids segregate into only one of the daughter cells, thus giving rise to plasmid free cells in the population. This event happens independent of what kind of plasmid the cell contains.</i></p>
+<p>X<sup>+</sup> is the cells with the plasmid.</p>
+<p>X<sup>-</sup> is the cells without the plasmid.</p>
+<p>u<sup>+</sup> is the specific cell growth rate of cells with the plasmid.</p>
+<p>u<sup>-</sup> is the specific cell growth rate of cells without the plasmid.</p>
+<p>p is the probability of a cell to lose a plasmid.</p>
+<br>
-<html><br></html>
+<p>We are trying to come up with a mathematical model for the relative populations of plasmid containing and plasmid free cells with respect to time using the growth curves that have been generated and the results from the fluorescence.</p>
-<html><br></html>
-[[Image:growthcurves.jpg|300px]]
+</html>
-<html><br></html>
+===Fluorescence Imaging of cells to check for directed plasmid loss===
-<p CLASS="justifyalign">
-<b><font color="#000">Fig 2.2:</font></b><i>Cells containing plasmids have a higher metabolic strain of synthesizing the proteins that are encoded by the plasmid, thus they tend to grow slower than the cells without any plasmids. The growth difference is pronounced especially during the exponential phase.</i></p>
-<html><br></html>
+This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how
-<html><br></html>
+# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium
+# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.
-<p CLASS="justifyalign">Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids.
+# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.
-Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.</p>
+# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.
-[[Image:expressionofgeneofinterest.jpg|500px]]
 <html><br></html>
-<p CLASS="justifyalign">
+[[Image:flourescenceimagingexp.jpg|700px]]
-<b><font color="#000">Fig 2.3:</font></b><i>In the initial state, all the cells bear a network of plasmids which can sustain each other under the influence of a particular selection pressure. In this state, the plasmids other than the one containing the gene of interest have repressors for the promoter that expresses the gene of interest in plasmid 1. As the cells are grown in a correct order of varying selection pressures, the cells lose plasmids directionally, thus leading to the expression or "unlocking" of the gene of interest.</i></p>
 <html><br></html>
+<html><b><font color="#000">Fig 6.3: </font></b><i>Experimental procedure for measuring the rate of plasmid loss by fluorescent imaging</i>
-===Circuit===
-<p CLASS="justifyalign">One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a highly regulated fashion. The plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or function in the cell. This will be the “unlocking” of the lock.</p>
-----
 <html><br></html>
-The grand idea of a 3 plasmid locking system revolved around constructing something like the below:
-<html><br></html>
+We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3.
-<html><br></html>
+# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol
+# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.
+# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol.
-[[Image:3-plasmid system.jpg|650px]]
-<html><br></html>
-<p CLASS="justifyalign">
+The expected behavior of the system is (listed in the same order as the above 3 conditions):
-<b><font color="#000">Fig 3.1:</font></b> <i>This is a general circuit for a 3 plasmid system which can show locking property with a code length of 2 units - Ab2->Ab1. This means that the cells need to be grown first in antibiotic medium 2 and then transferred to a medium containing antibiotic 1. It is easy to see that to maintain the whole system of 3 plasmids, the cells need to be grown in medium containing antibiotic 3.</i></p>
+# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.
+# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.
+# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.
-<html><br></html>
-<html><br></html>
-To which we thought we could fit in the following parts (the gene of interest is not included. It could be anything):
 <html><br></html>
-[[Image:constructs3plasmids.jpg|700px]]
+<html><b><font color="#000">Protocol: </font></b>
+<html><br></html><html><br></html>
+. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.
 <html><br></html>
+. Culture is grown for 4 hours or till the OD600 crosses 0.1.
-<b><font color="#000">Fig 3.2:</font></b><i>The 3 plasmid system.</i>
-----
 <html><br></html>
-Then we scaled it down and began to build constructs for a 2 plasmid system which works like this:
+. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).
 <html><br></html>
-[[Image:2-plasmid system.jpg|650px]]
 <html><br></html>
+Required inoculations are:
-<p CLASS="justifyalign">
+a. Broth 1 into tube with no antibiotic in the LB - tube 1
-<b><font color="#000">Fig 3.3:</font></b><i>This is a general circuit for a 2 plasmid system that can show locking property with a code length of 1 unit - which means the system is unlocked when it is grown in a medium containing antibiotic 1.</i></p>
 <html><br></html>
+b. Broth 1 into tube with Ampicillin in the LB - tube 2
 <html><br></html>
+c. Broth 2 into tube with no antibioti in the LB - tube 3
-To which we fit in parts from the registry to make it look like this:
 <html><br></html>
+d. Broth 2 into tube with Chloramphenicol in the LB - tube 4
-[[Image:constructs2plasmids.jpg|600px]]
 <html><br></html>
+e. Broth 3 into tube no antibiotic in the LB - tube 5
-<p CLASS="justifyalign">
-<b><font color="#000">Fig 3.4:</font></b><i>The 2 plasmid system.</i></p>
-----
 <html><br></html>
+f. Broth 3 into tube with Ampicillin in the LB - tube 6
-For the proof of concept experiments, we built the following constructs:
 <html><br></html>
+g. Broth 3 into tube with Chloramphenicol in the LB - tube 7
-[[Image:k272001.jpg]][http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]
 <html><br></html>
+h. Broth 3 into tube with both the antibiotics in the LB - tube 8
-<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of RFP in the transformed cells.</i>
 <html><br></html>
-[[Image:k272002.jpg]][http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]
 <html><br></html>
+. This culture is allowed to grow for 2.5 hours.
-<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of CFP in the transformed cells.</i>
 <html><br></html>
+. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.
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+. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes.
+. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.
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-===Expected behavior of the system ===
+Click [https://2009.igem.org/Team:IIT_Madras/Results#Fluorescent_Imaging here] to see the results of the fluorescence imaging.
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-<b><font color="#000">Fig 4.1:</font></b>[[Image:2plasmidcase-1.jpg|650px]]
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-''' Fig 4.2''':[[Image:2plasmidcase-2.jpg|650px]]
-==Work Plan==
-==Building the constructs==
-We have been following 3 different strategies to build our constructs.
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-)The 3A assembly
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-[[Image:3Aassembly.jpg|400px]]
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-)The 2A or Standard Assembly
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-[[Image:standardassembly.jpg|350px]]
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-)'''PCR based approach:''' We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.
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-This strategy saves a lot of time in cloning the parts together. All it takes is a PCR, a purification step, digestion and ligation. This strategy takes much less time in comparision with the traditional cloning steps using bacteria. Moreover, this process can be automated to ligate a huge number of parts together in a short period of time. It saves the labor of dealing with bacteria for amplifying the required insert after each ligation.
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-[[Image:PCR strategy.jpg|750px]]
-==Working with the construct==
-<p CLASS="justifyalign">We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus making the plasmids compete with each other in order to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.</p>
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-<p CLASS="justifyalign">The cotransformed cells will be grown in different antibiotic media (in our case - Ampicillin, Chloramphenicol and both Ampicillin and Chloramphenicol) and the loss of each plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the co-transformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with  both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.</p>
-[[Image:proof-of-concept.jpg|650px]]
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-<b><font color="#000">Fig 6:</font></b><i>The cotransformed cells when grown in different antibiotic media, show different fates. For instance, when the cells are grown in medium containing Ampicillin but not Chloramphenicol, the lack of selection pressure due to chloramphenicol on the cells leads to a gradual phasing out of those cells which contain both the plasmids. Only those with the Ampicillin selection marker eventually dominate the population.</i></p>
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-The constructs required to demonstrate the locking property are being built and will be tested once the construction is done.
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-===Experiments===
-====3A assembly====
-====Standard Assembly====
-====PCR based Approach====
-==Results==