Difference between revisions of "Team:Virginia/Results"

 
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                 With only 5 weeks of lab, there was a rush to verify sequences with restriction enzyme digests, build any of the preliminary parts, and visualize <i>E. coli</i> with BMCs in them. Unfortunately, the time limitation prevented us from reaching many of the steps we planned for in the last 10 months, but shown below are results from one major series of procedures we performed to produce BMCs in BL21(DE3) <i>E. coli</i>. With the generous help of Dr. Criswell at UVA, <b>we were able to successfully culture the cells, fixate them, and take pictures of 6 different samples using a Transmission Electron Microscope (TEM).</b>  
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                 With only 5 weeks of lab, there was a rush to verify sequences with restriction enzyme digests, build any of the preliminary parts, and visualize <i>E. coli</i> with BMCs in them. Unfortunately, the time limitation prevented us from succesfully completing most of the steps we planned for in the last 10 months, but shown below are results from one series of procedures we performed to produce BMCs in <i>E. coli</i> BL21(DE3). With the generous help of Dr. Criswell at UVA, <b>we were able to successfully culture the cells, fixate them, and take pictures of 6 different samples using a Transmission Electron Microscope (TEM).</b>  
 
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               The series of TEM Setup procedures are described in the Notebook page for 10/20 and 10/21. We transfected BL21(DE3) cells with with pdu-ABJKNU and pdu-ABJKNUT, and then prepared 5 plates as follows:  
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               The series of TEM Setup procedures are described in the Notebook page for 10/20 and 10/21. We transfected the  BL21(DE3) cells with with pdu-ABJKNU and pdu-ABJKNUT, and then prepared 5 plates as follows:  
 
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               Plates 1, 2, and 5 are pictured below and were the cultured cells we used for preparing TEM samples. As seen in Figure 1, the higher concentration of pdu-ABJKNU cells grew at a similar rate to the control cells and the diluted pduABJKNUT, which suggests that pdu-ABJKNU had a heavier metabolic load.
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               Plates 1, 2, and 5 are pictured below and were the cultured cells we used for preparing TEM samples. As seen in Figure 1, the higher concentration of pdu-ABJKNU cells grew at a similar rate to the control cells and the diluted pduABJKNUT.
 
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              <i><b>Fig 1: Plates for TEM</b> [A] shows the Untransformed BL21(DE3) control strain. [B] shows the Transformed pdu-ABKJNU cells on plate 2. [C] shows the Transformed pdu-ABJKNUT cells on plate 5.</i>
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               <b><i>E. coli</i> TEM Results Sample</b>: Top left is a sample from Culture 1 (Untransformed), top center is a sample from Culture 2 (Untransformed + IPTG), top right is a sample from Culture 3 (Transformed with pduABJKNU), bottom left is a sample from Culture 4 (Transformed with pduABJKNU + IPTG), bottom center is a sample from Culture 5 (Transformed with pduABJKNUT), and bottom right is a sample from Culture 6 (Transformed with pduABJKNUT + IPTG). Open each image in a new tab to view it at full scale. The whole collection of images is available <a href="https://static.igem.org/mediawiki/2020/e/e1/T--Virginia--media--TEM_Results.pdf">here</a>.
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               <b><i>Fig 2: E. coli under TEM</i></b> Top left is a sample from Culture 1 (Untransformed), top center is a sample from Culture 2 (Untransformed + IPTG), top right is a sample from Culture 3 (Transformed with pduABJKNU), bottom left is a sample from Culture 4 (Transformed with pduABJKNU + IPTG), bottom center is a sample from Culture 5 (Transformed with pduABJKNUT), and bottom right is a sample from Culture 6 (Transformed with pduABJKNUT + IPTG). Open each image in a new tab to view it at full scale. The whole collection of images is available <a href="https://static.igem.org/mediawiki/2020/e/e1/T--Virginia--media--TEM_Results.pdf">here</a>.
 
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Latest revision as of 03:32, 28 October 2020

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Results
Summary of Results
With only 5 weeks of lab, there was a rush to verify sequences with restriction enzyme digests, build any of the preliminary parts, and visualize E. coli with BMCs in them. Unfortunately, the time limitation prevented us from succesfully completing most of the steps we planned for in the last 10 months, but shown below are results from one series of procedures we performed to produce BMCs in E. coli BL21(DE3). With the generous help of Dr. Criswell at UVA, we were able to successfully culture the cells, fixate them, and take pictures of 6 different samples using a Transmission Electron Microscope (TEM).
TEM Set-Up
The series of TEM Setup procedures are described in the Notebook page for 10/20 and 10/21. We transfected the BL21(DE3) cells with with pdu-ABJKNU and pdu-ABJKNUT, and then prepared 5 plates as follows:
  1. BL21(DE3) control
  2. pdu-ABJKNU
  3. pdu-ABJKNU, diluted cell concentration
  4. pdu-ABJKNUT
  5. pdu-ABJKNUT, diluted cell concentration
Plates 1, 2, and 5 are pictured below and were the cultured cells we used for preparing TEM samples. As seen in Figure 1, the higher concentration of pdu-ABJKNU cells grew at a similar rate to the control cells and the diluted pduABJKNUT.
Fig 1: Plates for TEM [A] shows the Untransformed BL21(DE3) control strain. [B] shows the Transformed pdu-ABKJNU cells on plate 2. [C] shows the Transformed pdu-ABJKNUT cells on plate 5.

TEM Pictures
The following 6 cultures were fixated for TEM use as follows:
  1. BL21(DE3)
  2. BL21(DE3) + IPTG
  3. pdu-ABJKNU
  4. pdu-ABJKNU + IPTG
  5. pdu-ABJKNUT
  6. pdu-ABJKNUT + IPTG
A sample picture has been selected from each of the samples and is shown in Figure 2. We were not able to determine any significant difference between the samples, suggesting that there was a problem with the production of BMCs. The most likely reason for this is a deviation from the protocol we intended to follow due to a time limitation. Instead of allowing the samples to sit at room temperature overnight, we only allowed them to sit for a few hours, which led us to fix the cells before enough time for BMC formation had passed.

Fig 2: E. coli under TEM Top left is a sample from Culture 1 (Untransformed), top center is a sample from Culture 2 (Untransformed + IPTG), top right is a sample from Culture 3 (Transformed with pduABJKNU), bottom left is a sample from Culture 4 (Transformed with pduABJKNU + IPTG), bottom center is a sample from Culture 5 (Transformed with pduABJKNUT), and bottom right is a sample from Culture 6 (Transformed with pduABJKNUT + IPTG). Open each image in a new tab to view it at full scale. The whole collection of images is available here.


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