Difference between revisions of "Team:UMaryland/HokSok"

Revision as of 03:37, 19 September 2015

Purpose

When setting up our experimental design, we focused on answering three main questions:

1. Can Hok-Sok maintain a plasmid over many generations? If so, how does its performance compare to classical antibiotic pressure systems?
2. Does the Hok-Sok cassette restrict the growth of the bacteria it resides in?
3. Do proximity effects occur between Hok-Sok and a neighboring expression construct? Alternatively, does the activity of Hok-Sok affect expression of a protein whose coding region is near the construct?

4. Can this cell living with Hok-Sok stay happy?

The ability to use plasmids as vectors to introduce genes of interest in E. coli is one of the most essential bioengineering tools. However, one of the limitations of transforming a bacterium with a plasmid, is that the organism will eventually eject the plasmid over time. To counter this, scientists add a positive selective pressure on E. coli to retain plasmids carrying resistance genes through the use of antibiotics. While this technique has proven to be reliable and effective, there are many limitations. The prevalent use of antibiotics both for medical and agricultural purposes has rapidly increased the number of pathogens that harbor antibiotic resistant genes. As a result, there is a pressing need to find an alternative to antibiotic use for plasmid maintenance to prevent the spread of antibiotic resistant genes. Many synthetic biology projects which focus of solving health or environmental issues are confined to the lab because of these limitations. The University of Maryland iGEM team seeks to solve this problem by developing an alternative plasmid maintenance system that should liberate other iGEM teams from the dependance on antibiotic usage. We hypothesized that Hok-Sok, our plasmid maintenance system, could maintain recombinant plasmids, as it does natural ones. We also hypothesized that Hok-Sok would have a slight negative effect on bacterial growth rate, in line with other alternative maintenance systems such as sRNBC (K817015), as well as the amount of protein expression due to competing parallel promoters. In order to answer these questions, we set up a variety of testing procedures, as shown below.

Section Summary

1. We wanted to answer three main questions concerning the ability of Hok-Sok to maintain a plasmid.
2. We hypothesized that Hok-Sok would be able to replicate its natural maintenance ability for a recombinant plasmid.
3. We devised tests to measure maintenance over time.

Hok/Sok Construct

Prior to experimentation, we had to insert the Hok-Sok construct into pSB1C3 in order to make it a BioBrick. We originally planned to PCR amplify the cassette out of the R1 plasmid of E. coli, but we were unable to find an suitable wild-type strain that was easily available. Instead, we turned to synthesizing the construct as a gBlock from IDT. As a 580 bp dsDNA fragment, it was suitable for addition via Gibson Assembly into pSB1C3. After subsequent transformation, miniprep, and confirmation sequencing, we had the first piece of our testing puzzle.

Proof that at least some of our cloning worked

Section Summary

1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R1 plasmid of E. coli.
2. g-Block of Hok-Sok sequence ordered from Integrated DNA Technologies, then assembled into pSB1C3 using the Gibson Assembly method.

Fluorescence Studies

In order to determine if Hok/Sok was capable of maintaining a plasmid without antibiotic pressure, we decided to use a visual reporter gene to quantify the ability of Hok/Sok to maintain plasmids over many generations. We decided to use a RFP along with a degradation tag as the reporter gene. The most suitable candidate was an unstable LVA-tagged RFP that has a half-life of 1 hour. The shorter half life allows for more frequent measurements of protein production that would not aggregate over time. Therefore we combined a constitutive promoter and RBS to the LVA-tagged RFP through 3A assembly. We transformed this construct to E. coli Dh5 alpha to confirm the effectiveness of this reporter gene and its expression through increased fluorescence. Afterwords we ordered a g-block of our Hok/Sok+reporter construct. The expression of this reporter gene is proportional to plasmid number. Therefore, we concluded that if the cells containing a plasmid with both Hok/Sok and reporter gene could maintain fluorescence over many generations without the positive pressure of antibiotics compared to our controls, Hok/Sok can be used as a viable plasmid maintenance system. We transformed this Biobrick onto both Dh5 alpha and BL21 strains for testing. We tested fluorescence of 3 biological and 3 technical replicates of the 5 groups listed below using a microplate reader. The 5 groups and their replicates were picked off of plates and incubated in 5 mLs of LB in culture tubes. A 1000x chloramphenicol concentration was added to groups A, C, and D. There was no chloramphenicol added to groups B and E. After 20 hours, 200 uL of each overnight culture was transferred onto a 96 well plate and the fluorescence data was recorded. 4 hours later, 50 uL of this overnight was inoculated in a new culture tube containing 5 mL of LB. These new cultures were the new generation, and they were incubated for 20 more hours for more testing. This process was repeated for several generations.

For our first testing method to see if Hok-Sok was capable of maintaining a plasmid, we wanted to measure how RFP fluorescence was retained over many generations in two E. coli strains: BL21 and DH5α.

We chose unstable Red Fluorescent Protein (RFP) as a marker for all our test groups to represent whether or not the inserted plasmid is still present in the bacteria. If the plasmid is maintained, the RFP is expressed and the overall fluorescence of the culture is greater. In contrast, if the bacteria does not feel enough pressure to keep the plasmid and ejects it, the measured fluorescence is on the lower end. From this data, we can gather whether or not the maintenance system is effective in preserving a plasmid in bacteria that is not beneficial to its survival, such as the aforementioned RFP. The instability of the RFP is pertinent; the half-life of the proteins is shorter than a stable protein, therefore we can tell in real-time, or at least more so, whether or not the plasmids are present. The RFP degrades and unless the plasmid is maintained, the fluorescence in the cells actively declines. We originally used BL21 strain of E.coli because it is known to be the best for testing because the cell lacks proteases; the protein expression is optimal because the proteins are not digested by the enzymes. After testing BL21, we transitioned to the DH5a strain of E. coli because the cells lack recombinase.

A. Constitutive unstable RFP grown with chloramphenicol (33 µg/mL) in media (+ Control)
B. Constitutive unstable RFP grown without chloramphenicol
C. Unstable RFP without promoter with chloramphenicol (- Control)
D. Hok-Sok + Constitutive unstable RFP grown with chloramphenicol
E. Hok-Sok + Constitutive unstable RFP grown without chloramphenicol

A. 200 µL of undiluted culture was pipetted into each well.
B. Excitation wavelength was 555 nm. Emission wavelength was 584 nm.

Section Summary

1. Hok-Sok was coupled to a constitutive generator of unstable RFP to form a larger composite part.
2. In order to test the ability of Hok-Sok to maintain protein expression, five sets of cultures were grown in LB media for fluorescence studies.
3. Cultures were grown for 20 hours overnight prior to fluorescence measurements using a plate reader.

@@ Line 163: / Line 163: @@
 <p style="font-size:24px">In order to determine if Hok/Sok was capable of maintaining a plasmid without antibiotic pressure, we decided to use a visual reporter gene to quantify the ability of Hok/Sok to maintain plasmids over many generations. We decided to use a RFP along with a degradation tag as the reporter gene. The most suitable candidate was an unstable LVA-tagged RFP that has a half-life of 1 hour. The shorter half life allows for more frequent measurements of protein production that would not aggregate over time. Therefore we combined a constitutive promoter and RBS to the LVA-tagged RFP through 3A assembly. We transformed this construct to E. coli Dh5 alpha to confirm the effectiveness of this reporter gene and its expression through increased fluorescence. Afterwords we ordered a g-block of our Hok/Sok+reporter construct. The expression of this reporter gene is proportional to plasmid number. Therefore, we concluded that if the cells containing a plasmid with both Hok/Sok and reporter gene could maintain fluorescence over many generations without the positive pressure of antibiotics compared to our controls, Hok/Sok can be used as a viable plasmid maintenance system. We transformed this Biobrick onto both Dh5 alpha and BL21 strains for testing. We tested fluorescence of 3 biological and 3 technical replicates of the 5 groups listed below using a microplate reader. The 5 groups and their replicates were picked off of plates and incubated in 5 mLs of LB in culture tubes. A 1000x chloramphenicol concentration was added to groups A, C, and D. There was no chloramphenicol added to groups B and E. After 20 hours, 200 uL of each overnight culture was transferred onto a 96 well plate and the fluorescence data was recorded. 4 hours later, 50 uL of this overnight was inoculated in a new culture tube containing 5 mL of LB. These new cultures were the new generation, and they were incubated for 20 more hours for more testing. This process was repeated for several generations. </li>
 <li> For our first testing method to see if Hok-Sok was capable of maintaining a plasmid, we wanted to measure how RFP fluorescence was retained over many generations in two <i>E. coli</i> strains: BL21 and DH5α.</li>
+<p style="font-size:24px">We chose unstable Red Fluorescent Protein (RFP) as a marker for all our test groups to represent whether or not the inserted plasmid is still present in the bacteria. If the plasmid is maintained, the RFP is expressed and the overall fluorescence of the culture is greater. In contrast, if the bacteria does not feel enough pressure to keep the plasmid and ejects it, the measured fluorescence is on the lower end. From this data, we can gather whether or not the maintenance system is effective in preserving a plasmid in bacteria that is not beneficial to its survival, such as the aforementioned RFP.
+The instability of the RFP is pertinent; the half-life of the proteins is shorter than a stable protein, therefore we can tell in real-time, or at least more so, whether or not the plasmids are present. The RFP degrades and unless the plasmid is maintained, the fluorescence in the cells actively declines.
+We originally used BL21 strain of E.coli because it is known to be the best for testing because the cell lacks proteases; the protein expression is optimal because the proteins are not digested by the enzymes. After testing BL21, we transitioned to the DH5a strain of E. coli because the cells lack recombinase.
 <ol>
 <li>A. Constitutive unstable RFP grown <b>with</b> chloramphenicol (33 µg/mL) in media <b>(+ Control)</b></li>