Team:Berkeley/Project/Self Lysis

From 2010.igem.org

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Latest revision as of 21:24, 27 October 2010

Overview

The Self Lysis Device acts to degrade the bacterial membrane (inner membrane, peptidoglycan layer and outer membrane). In the overall delivery scheme, if the timing works out so that self-lysis occurs after the bacteria has been consumed by the Choanoflagellate, the payload will be released into the vesicle.

The Construct

Pbad promoter: allows for fast, controlled induction, which allows us to better control the timing of lysis. Also, since this promoter is only induced by an exogenous molecule, it helps address biosafety concerns.

Bacteriocin Releasing Protein (BRP): degrades the outer membrane

Lysozyme: degrades the peptidoglycan layer

Holin: creates pores in the inner membrane
Anti-holin: threshold-gating system

(for more complete descriptions, see 2008 Berkeley iGEM team's wiki)

Our Story: From 2008 to 2010

Our project involved using a modified version of the 2008 Berkeley iGEM construct in an entirely new, innovative setting. The 2008 team hoped that Self-lysis would be a means of product purification and characterized the part in a standard E. coli media, Luria Broth (LB), as outlined here. In the Anderson lab, the construct was modified to include the BRP protein and has been extensively tested as a means of delivery in Mammalian cells. We, the 2010 Berkeley iGEM team, are using this device in an entirely new setting: the single celled, lower metazoans called Choanoflagellates.

The Magnesium Promoter
Our team started out by using a version of the Self-lysis device that was under a Magnesium promoter, which turns on in low levels of Mg. This construct has been used extensively in the Anderson lab for projects in Mammalian delivery, since it is a means for the E. coli to detect when it has been encased in a phagocytic vesicle and thus trigger self-lysis in the appropriate setting. However, since this promoter takes about 4 hours to turn on and we soon realized that Choanoflagellates eat and digest bacteria along with its payload in about an hour, this promoter is much too slow for our delivery scheme. Thus, we decided to switch out the Magnesium promoter for a Pbad promoter, which allows Self-lysis to occur within an hour after induction.

Media Characterization
But switching out a promoter didn't end our battle to apply this construct to a new system: we had to find a media that would be an appropriate environment for both the E. coli and Choanoflagellates. Berkeley 2008 characterized the part in LB, but Choanoflagellates are sluggish and generally unhealthy in LB. Thus, we had to find another media in which to perform our delivery scheme.

Characterization in new medias

The 2008 team already characterized the device in LB, however, choanoflagellates are not healthy in LB media. Since we are applying the device to a new setting, we needed to characterize it in new conditions. Our challenge was to find a media that would both allow lysis to occur and keep the Choanoflagellates happy and healthy.

Medias Tested

Luria Broth (LB): Positive Control, used to confirm the results of the iGEM 2008 team and to make sure the assembly with the Vesicle Buster Device didn't disrupt lysis, since it tends to be a fairly tempermental part.
Terrific Broth (TB): Used to grow up our payload delivery device and payload containing bacteria to a high concentration before feeding them to the Choanos. E. coli flourish in TB.
Cereal Grass Media (CGM): Developed by the King lab to culture Choanos.
Artificial Sea Water (ASW): Sterilized salt water that we use to culture our line of Choanos.
Different ASW:LB ratios
Spring water and wheat seed medium (used to culture many fresh water eukaryotes)
Different Spring water:LB ratios

Additional information about CGM and ASW can be found [http://www.choano.org/wiki/Choano_Media here].

Testing lysis

Self-lysis can be assayed by measuring changes in optical density, or changes in the opaqueness of a solution. As lysis occurs, optical density decreases, meaning the solution of bacteria becomes more translucent.

On a macro-scale, here's what you'd expect to see from a working self-lysis device after incubating the solutions for 1 hour at 37degC with shaking:

We used a TECAN machine to measure the change in optical density over time. The following data points are averages from several runs on colonies from the same transformation. The data has been normalized by dividing each OD data point by the initial measure of OD and shows error bars based on the standard deviation. This run shows our comparison of lysis in CGM media to lysis in LB:

The above data shows that Lysis does indeed take place in CGM media, even if it's not as robust as in LB. Similar tests showed that TB is the best media for lysis, followed by LB, CGM, high ratios of LB:ASW, low ratios of LB: ASW, and ASW. Also, macroscopic tests indicate that lysis occurs in spring water:LB ratios as high as 80:20.

However, this data wasn't enough to reveal what media should be used to run our delivery assay. We needed to see which of these medias supported Choanoflagellate health as well.

Observing Choanoflagellate health at 37C

We needed to ensure that while our device went into action, the choanoflagellates were not going to be exposed to environmental conditions that could damage them.

Since our E. coli operated optimally at 37 degrees Celsius, we tested whether choanoflagellates could be exposed to this temperature (about 10 degrees higher than their preferred temperature) for a few hours while our device functioned. We split a culture of choanoflagellates (approaching stationary phase) into two equal cultures. We placed one in a room temperature drawer and one in the 37 degree incubator. We removed 10uL samples and counted the cell concentration at 4 and 25 hours. In the graphs below, you can see that after 4 hours, there was little effect. In fact, there were slightly more choanos in the warmer culture and they were much more active, meaning they swam around faster. After 25 hours at the specified temperatures, we had begun loosing cells in the 37 degree culture, as can be seen in the figure below. However, this will not be a problem, as our device only needs about two hours to reach maximum delivery.

We were also cautious about putting choanoflagellates into bacterial media. Choanos, as mentioned before, prefer to live in sea water or sea water-based medias, such as CGM. They prefer a much higher solute content than bacteria media can offer. When transferred the choanos into LB, our concerns were verified. The choanos stopped swimming, and merely twitched weakly on the plate bottom.

The best media for delivery

This table sums up the results of our lysis and Choano health assays:

The best media proved to be CGM, Cereal Grass Media, because it both kept the Choanoflagellates healthy and allowed lysis to occur. Thus, although we continued to maintain our culture of Choanoflagellates in ASW and grow up our E. coli in TB, we performed all of our payload delivery assays in CGM.

@@ Line 38: / Line 38: @@
 ''Media Characterization''<br>
-But switching out a promoter didn't end our battle to apply this construct to a new system: we had to find a media that would be an appropriate environment for the E. coli and Choanoflagellates. Berkeley 2008 characterized the part in LB, but Choanoflagellates are sluggish and generally unhealthy in LB. Thus, we had to find another media in which to perform our delivery scheme.
+But switching out a promoter didn't end our battle to apply this construct to a new system: we had to find a media that would be an appropriate environment for both the E. coli and Choanoflagellates. Berkeley 2008 characterized the part in LB, but Choanoflagellates are sluggish and generally unhealthy in LB. Thus, we had to find another media in which to perform our delivery scheme.
@@ Line 44: / Line 44: @@
 <font size="5">'''Characterization in new medias'''</font>
-The 2008 team already characterized the device in LB, as shown here (insert link). However, since we are applying the device in a brand new setting, we needed to characterize it in that new setting. Our challenge was to find a media that would both allow lysis to occur and keep the Choanoflagellates happy and healthy.
+The 2008 team already characterized the device in LB, however, choanoflagellates are not healthy in LB media. Since we are applying the device to a new setting, we needed to characterize it in new conditions. Our challenge was to find a media that would both allow lysis to occur and keep the Choanoflagellates happy and healthy.
 <font size=3>'''Medias Tested'''</font>
@@ Line 53: / Line 53: @@
 *Artificial Sea Water (ASW): Sterilized salt water that we use to culture our line of Choanos.
 *Different ASW:LB ratios
+*Spring water and wheat seed medium (used to culture many fresh water eukaryotes)
+*Different Spring water:LB ratios
 Additional information about CGM and ASW can be found [http://www.choano.org/wiki/Choano_Media here].
@@ Line 60: / Line 62: @@
 Self-lysis can be assayed by measuring changes in optical density, or changes in the opaqueness of a solution. As lysis occurs, optical density decreases, meaning the solution of bacteria becomes more translucent.
-On a macro-scale, here's what you'd expect to see from a working self-lysis device:
+On a macro-scale, here's what you'd expect to see from a working self-lysis device after incubating the solutions for 1 hour at 37degC with shaking:
 <center>[[Image:macro self lysis.png | 200px]]</center>
-We used a TECAN machine to measure the change in optical density over time. The following data points are averages from several runs on colonies from the same transformation. The data has been normalized (insert details) and show error bars (insert details!).
+We used a TECAN machine to measure the change in optical density over time. The following data points are averages from several runs on colonies from the same transformation. The data has been normalized by dividing each OD data point by the initial measure of OD and shows error bars based on the standard deviation. This run shows our comparison of lysis in CGM media to lysis in LB:
-<font size=10>TECAN DATA</font>
+[[Image:tecan old.png | 500px]]
-Thus, TB is the best media for lysis, followed by LB, CGM3, high ratios of LB:ASW, low ratios of LB: ASW, and ASW.
+The above data shows that Lysis does indeed take place in CGM media, even if it's not as robust as in LB. Similar tests showed that TB is the best media for lysis, followed by LB, CGM, high ratios of LB:ASW, low ratios of LB: ASW, and ASW. Also, macroscopic tests indicate that lysis occurs in spring water:LB ratios as high as 80:20.
+However, this data wasn't enough to reveal what media should be used to run our delivery assay. We needed to see which of these medias supported Choanoflagellate health as well.
-<font size=3>'''Observing Choanoflagellate health'''</font>
-We observed the Choanoflagellates after incubating them in TB and LB, and they appeared to be very sluggish, which is a sign that they are unhealthy.
+<font size=5>'''Observing Choanoflagellate health at 37C'''</font>
-Choanos at 37deg?? Conor's data?
+We needed to ensure that while our device went into action, the choanoflagellates were not going to be exposed to environmental conditions that could damage them.
-<font size=10>CONOR's DATA</font>
+Since our ''E. coli'' operated optimally at 37 degrees Celsius, we tested whether choanoflagellates could be exposed to this temperature (about 10 degrees higher than their preferred temperature) for a few hours while our device functioned. We split a culture of choanoflagellates (approaching stationary phase) into two equal cultures. We placed one in a room temperature drawer and one in the 37 degree incubator. We removed 10uL samples and counted the cell concentration at 4 and 25 hours. In the graphs below, you can see that after 4 hours, there was little effect. In fact, there were slightly more choanos in the warmer culture and they were much more active, meaning they swam around faster.  After 25 hours at the specified temperatures, we had begun loosing cells in the 37 degree culture, as can be seen in the figure below. However, this will not be a problem, as our device only needs about two hours to reach maximum delivery.
+[[image:Choano4.png|300px]]               [[image:Choano25.png|300px]]
-<font size=3>'''Conclusion'''</font>
+<br>
+We were also cautious about putting choanoflagellates into bacterial media. Choanos, as mentioned before, prefer to live in sea water or sea water-based medias, such as CGM. They prefer a much higher solute content than bacteria media can offer. When transferred the choanos into LB, our concerns were verified. The choanos stopped swimming, and merely twitched weakly on the plate bottom.
+<font size=5>'''The best media for delivery'''</font>
 This table sums up the results of our lysis and Choano health assays:
 [[Image:media assay chart.png | 200px]]
-The best media proved to be CGM, Cereal Grass Media, because it both kept the Choanoflagellates healthy and allowed lysis to occur. Thus, although we continued to culture our Choanoflagellates in ASW and grow up our E. coli in TB, we performed all of our payload delivery assays in CGM.
+The best media proved to be CGM, Cereal Grass Media, because it both kept the Choanoflagellates healthy and allowed lysis to occur. Thus, although we continued to maintain our culture of Choanoflagellates in ASW and grow up our E. coli in TB, we performed all of our payload delivery assays in CGM.
-Self-lysis Device
-To break through the E. coli’s inner and outer cell wall, we used the self-lysis device derived from the 2008 UC Berkeley iGEM team (registry name and picture of 2008 part). There were, however, several challenges we needed to overcome before the device could be applied in a choanoflagellate. First, the lysis device had to be inducible by an exogenous molecule. For practical reasons we needed to be able to control when self-lysis occurred and for biosafety reasons self-lysis could not occur outside of a laboratory culture. For example, we initially constructed the self-lysis under a magnesium based promoter but that construct was not used because magnesium is commonly found in sea water and mammalian cells and would lead to lysis in undesirable and potentially dangerous situations. By putting the device under the control of an arabinose-induced promoter, Pbad, we were able to induce lysis only when desired and prevent incidental lysis. Moreover, self-lysis had to be fast acting: the bacteria had to lyse itself after it was ingested by the choanoflagellate but before it was digested by the choanoflagellate. The 2008 part took closer to five hours to lyse but we estimated that choanoflagellates digestion takes only one to two hours; therefore we needed a faster acting device. To accomplish this we added BRB to the original construct. BRB degrades the inner cell membrane. With this addition, we were able to have lysis occur within an hour after induction.
-Although the self-lysis device must be under control of an exogenous inducer, self lysis must also occur in the choanoflagellates culture. In the in vitro assay for self-lysis, E. coli were grown and induced in TB media. However, choanoflagellates cannot survive in TB or LB media. Similarly, an in vitro assay showed that self-lysis failed to occur when the bacteria were put in the artificial sea water used to culture choanoflagellates. We found a compromise between the health of choanoflagellates and the activity of the self-lysis device by using Choano Growth Media 3 (CGM3). As shown in the graph below, where a decrease in optical density indicates successful lysis, the bacteria were able to lyse themselves in CMG3 media almost as well as they did in TB.