Difference between revisions of "Team:UMaryland/Results"

Latest revision as of 04:00, 19 September 2015

Plating Tests - Counting Colonies to Confirm Chloramphenicol Resistance

Over time, all cultures appeared to maintain antibiotic resistance, except for the negative control, which experienced no pressure during its growth. The following figures demonstrate the presence of resistant colonies over many hundreds of bacterial generations. Most importantly, the Hok-Sok culture grown without antibiotic was shown to maintain chloramphenicol resistance. This sets up one of our major conclusions: the Hok-Sok cassette is capable of maintaining a recombinant plasmid for an extended period of time.

Negative Control (No Pressure)

Figure 1. Negative control was K1783002 (constitutive unstable RFP) grown in media without chloramphenicol. After 2-3 days, plates demonstrate fewer than 10 colonies per plate, suggesting that antibiotic resistance is now only minimally present in the culture.

Chloramphenicol Pressure Only

Figure 2. K1783002 (constitutive unstable RFP) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Chlor + Hok-Sok Pressure

Figure 3. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Hok-Sok Pressure Only

Figure 4. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media without chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Growth Curve

Figure 5. Growth curves of parallel cultures grown with and without chloramphenicol. The presence of Hok-Sok on the inserted plasmid does not appear to have a major effect on the bacterial growth rate. This is in line with our qualitative observations, where we do not observe any major difference in cell density during plating or fluorescence studies.

Fluorescence Studies

Our fluorescence studies supported the findings of our plating tests. We were able to observe that fluorescence was rapidly lost in the negative control. This was expected due to plating tests demonstrating the loss of plasmid from that group. We were pleasantly surprised to observe that Hok-Sok was able to maintain fluorescence for a longer period of time than typical chloramphenicol pressure.

DH5α Cell Line

Figure 6. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in DH5α cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is initially high, but rapidly diminishes over time.

Figure 7. K1783002 (Constitutive unstable RFP) in DH5α cells grown in media without antibiotic. Fluorescence is initially low and rapidly diminishes over time. This result supports the notion that cells grown without any pressure quickly lose their plasmids.

Figure 8. K1783003 (Hok-Sok+Constitutive unstable RFP) in DH5α cells grown in media with antibiotic. Fluorescence is initially moderate and diminishes over time. The use of two maintenance systems does not appear to better maintain fluorescence levels than chloramphenicol alone. We suggest that the proximity of the Hok-Sok cassette to the RFP construct somewhat represses expression of RFP due to promoter interference, leading to a lower initial reading.

Figure 9. K1783003 (Hok-Sok+Constitutive unstable RFP) in DH5α cells grown in media without antibiotic. Fluorescence is initially moderate, but remains relatively constant over time, a one-time spike notwithstanding.

Figure 10. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is variable throughout testing suggesting an uncorrected factor influencing results. Note: Our initial tests using the BL21 cell line were inconclusive due the need to calibrate our testing protocol.

Figure 11. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media without antibiotic.

Figure 12. Fluorescence measurements of K1783003 (Hok-Sok+Constitutive unstable RFP) in BL21 cells grown in media with antibiotic.

Figure 13. Fluorescence measurements of K1783003 (Hok-Sok+Constitutive unstable RFP) in BL21 cells grown in media without antibiotic.

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 <p><img src = "https://static.igem.org/mediawiki/2015/c/c1/UMDBL21A.png"></p>
-<p style = "font-size:18px">Figure 10. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is variable throughout testing suggesting an uncorrected factor influencing results. Note: Our initial tests using the BL21 cell line were inconclusive due to a variety of factors, including the need to calibrate our testing protocol. We are including them here to demonstrate the higher level of RFP fluorescence in a cell line engineered to express proteins well.</p>
+<p style = "font-size:18px">Figure 10. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is variable throughout testing suggesting an uncorrected factor influencing results. Note: Our initial tests using the BL21 cell line were inconclusive due the need to calibrate our testing protocol.</p>
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 <p style="font-size:48px;text-align:center;font-family:Verdana, Geneva, sans-serif;"><b>Sequence Analysis</b></p>
-<p style = "font-size:24px">Gels generally show that plasmids are kept whenever a form of pressure is placed on the cell. Why then, is RFP not being expressed? Our sequencing results showed random mutations in the promoter and coding region of the RFP construct. This is a valuable lesson that, with any BioBrick construct, mutations and evolution is inevitable. However, plasmids that were maintained with Hok-Sok alone (no chloramphenicol) did not display mutations in the RFP construct. The large difference in protein expression over multiple days, as shown by our fluorescence and plating tests, suggests to us that the presence of Hok-Sok, combined with the absence of chloramphenicol pressure, is putting a smaller evolutionary pressure on the  bacterium.</p>
+<img src = "https://static.igem.org/mediawiki/2015/thumb/1/19/UMDHS2gel.png/666px-UMDHS2gel.png.jpeg">
+<p style = "font-size:24px">Top right bands of above gel are of negative control in DH5-alpha. They demonstrate that the cells no longer have the plasmid of interest. Gels generally show that plasmids are kept whenever a form of pressure is placed on the cell. Why then, is RFP not being expressed? Our sequencing results showed random mutations in the promoter and coding region of the RFP construct. This is a valuable lesson that, with any BioBrick construct, mutations and evolution is inevitable. However, plasmids that were maintained with Hok-Sok alone (no chloramphenicol) did not display mutations in the RFP construct. The large difference in protein expression over multiple days, as shown by our fluorescence and plating tests, suggests to us that the presence of Hok-Sok, combined with the absence of chloramphenicol pressure, is putting a smaller evolutionary pressure on the  bacterium.</p>
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 <p style="font-size:48px;text-align:center;font-family:Verdana, Geneva, sans-serif;">Conclusions and Future Plans
+<p style = "font-size:32px">Conclusion</p>
+<p style = "font-size:24px">Through our plating tests and fluorescence measurements, we were able to successfully observe the plasmid maintenance ability of the Hok-Sok system. In comparison to a negative control grown without chloramphenicol pressure, cells containing Hok-Sok were able to maintain their antibiotic resistance for a significant period of time, as shown from our plating tests. In addition, we noted the ability of Hok-Sok had a negative effect on protein production, as shown from our fluorescence study. However, this level of production was consistent over a long period of time, contrasting with traditional pressure systems where mutations were able to build up quickly, shutting down production of RFP, which has no biological usefulness for the cell. We thus suggest that the downregulation of RFP production via Hok-Sok is capable of decreasing the evolutionary pressure against the removal on non-essential genes. This effect will be further studied in later experiments, where the distance between the hok-sok cassette and a gene of interest is lengthened. Overall, we suggest use of the Hok-Sok system as an effective method for internal plasmid maintenance without the use of antibiotics.</p>
 <p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">Considerations