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Team:BostonU/Data
From 2014.igem.org
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| - | <tr><td scope="col" colspan="2" >Below | + | <tr><td scope="col" colspan="2" >Below are the micrographs team WPI-Worcester took for us. They used confocal microscopy to view cells containing tandem promoter + RFP testing constructs. These constructs were also tested using flow cytometry as described above. For details on experimental setup, click <a href="https://2014.igem.org/Team:BostonU/Collaborations">here</a>. Both constructs expressed RFP for 100mM arabinose (on state) and did not express RFP for 1mM. While we did not test 100mM in our previous flow experiment, the confocal microscopy shows the same trend that we would expect for arabinose. For atc, the confocal experiment differed from our flow cytometry results because we expected an on state for 1000 ng/mL. This will require further investigation and a repeat experiment. It is possible that we mixed up the media that we gave to WPI. |
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<td scope="col"><img src="https://static.igem.org/mediawiki/2014/4/47/TBWPICollab.png" width="450" style="float:right" style= "margin-left:10px"></td> | <td scope="col"><img src="https://static.igem.org/mediawiki/2014/4/47/TBWPICollab.png" width="450" style="float:right" style= "margin-left:10px"></td> | ||
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<tr><td scope="col"><capt>Figure 5: Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pTet+pBAD+ RFP</capt></td> | <tr><td scope="col"><capt>Figure 5: Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pTet+pBAD+ RFP</capt></td> | ||
Revision as of 22:28, 16 October 2014
As a measurement team, we completed the Interlab Study. For more information about our Interlab Study results, please refer to our Interlab Study
page.
Flow Cytometry Data
pTet-pBad RFP Characterization with atc and arabinose | |
| For the first tandem promoter flow cytometry experiment, we tested pBad-pTet-BCD2-E1010m-B0015 and pTet-pBad-BCD2-E1010m-B0015. We planned on inducing each promoter separately with it's corresponding small molecule (either arabinose or atc) and also planned on inducing both together. We followed the flow cytometry workflow. By growing the constructs in different concentrations of media, we hoped to see RFP fluorescence increase as the small molecule concentration increased. For each concentration, we also had a negative. We also ran controls including: J04B2RM (RFP Positive), J04B2GM (GFP Positive), COXGR, COXRG, and DH5alpha. For an explanation of how we chose our controls, please refer to our Software Tools page.The pTet-pBad graph turned out the way we expected and showed the anticipated function. For the 5,000 and 10,000 ng/ul atc concentrations (for both graphs), the cells died because of the high concentrations. This is why the graph dropped rapidly. |  |
pBad-pTet RFP Characterization with atc and arabinose | |
| For the pBad-pTet construct, we used the same controls as mentioned above. According to literature, the pTet-pBad construct has not previously functioned as suspected. This was possibly due to position-dependence interference [1]. Conversely, our flow cytometer data showed that pTet-pBad had a greater range of fluorescence than pBad-pTet. We will need to do further investigating to find out why this was the case. Unlike results in the literature, our pTet-pBad construct worked well, but the pBad-pTet didn't show anticipated function. We are predicting that the arabinose concentrations were too low for this experiment and the atc concentrations were too high. We are planning to run another flow experiment before the jamboree with new small molecule concentrations. We hope that this will improve function (fluorescence expression) and reduce the error bars. |  |
Testing of p15A origin of replication | |
| The similarities in fluorescence between high- and medium-copy plasmids were not expected, as only approximately 10-25 plasmids should be present per cell with the p15A origin in contrast with the pMB1 origin, which allows for over 100 plasmids per cell. The Phase 1 cloning strategy must be modified to ensure proper incorporation of the p15A origins by the cells to see the predicted change in fluorescence. |  |
Collaboration with Team WPI-Worcester | |
| This data is a result of our collaboration with Team WPI-Worcester. They gave us two copies of the same construct, in different antibiotic resistant backbones. This construct expresses BclA-YFP , a cell surface targeted protein that expresses YFP on the cell surface. Our intention was to compare BclA expression with expression of our internal YFP control (J23104+BCD2+YFP+B0015). As evident from Figure 4, the Internal YFP has an expression of over 2 * 10^4 MEFLs, while none of the BclA constructs have expression more than 10^4 MEFLs. This data is, however, inconclusive as the internal YFP is in a Kanamycin resistant backbone and as we have shown, different backbones can lead to greatly varied data. |  |
| Below are the micrographs team WPI-Worcester took for us. They used confocal microscopy to view cells containing tandem promoter + RFP testing constructs. These constructs were also tested using flow cytometry as described above. For details on experimental setup, click here. Both constructs expressed RFP for 100mM arabinose (on state) and did not express RFP for 1mM. While we did not test 100mM in our previous flow experiment, the confocal microscopy shows the same trend that we would expect for arabinose. For atc, the confocal experiment differed from our flow cytometry results because we expected an on state for 1000 ng/mL. This will require further investigation and a repeat experiment. It is possible that we mixed up the media that we gave to WPI. | |
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|
Fusion Protein GFP Expression | |
| Though we were unable to successfully clone the testing constructs listed here, we tested the single transcriptional units with GFP and YFP fusion proteins to check whether there was any protein expression at all. It was hypothesized that because the two heavy gene sequences in a fusion protein are very close to each other, GFP expression wouldn't be as high as it would in a single GFP control. The hypothesis was accepted based on the results on Figure 7. However, it should be noted that the fluorescence for both tetR+GFP and araC+GFP is not greatly different from that for just GFP. This cannot be used to conclude whether the fusion proteins affect regulator function. For that, we will need to test the testing constructs designed. We should be able to present that data at the Jamboree. |  |
Eugene
Raven
SBOL
Primer Design for Tandem Promoters and Repressor Genes
| Device Name | Forward Primer | Sequence | Reverse Primer | Sequence |
|---|---|---|---|---|
| BetI_CD | BetI_For_C | ATGAAGACGTAATGGTGCCGAAACTGGGTATGCAGAGC | BetI_Rev_D | ACGAAGACCTACCTTTAATCGGTCGGCAGATGCTGGGT |
| PhlF_CD | PhlF_For_C | ATGAAGACGTAATGATGGCACGTACCCCGAGCCGTAGC | PhlF_Rev_D | ACGAAGACCTACCTTTAACGCTGTGTACCCGGACAAAC |
| BM3R1_CD | BM3R1_For_C | ATGAAGACGTAATGATGGAAAGCACCCCGACCAAACAG | BM3R1_Rev_D | ACGAAGACCTACCTTTAGCTCTGACGGCTCAGTGCTGC |
| LmrA_CD | LmrA_For_C | ATGAAGACGTAATGATGAGCTATGGTGATAGCCGTGAA | LmrA_Rev_D | ACGAAGACCTACCTTTAACGTTTCAGCAGATCCGGAAT |
| SrpR_CD | SrpR_For_C | ATGAAGACGTAATGATGGCACGTAAAACCGCAGCAGAA | SrpR_Rev_D | ACGAAGACCTACCTTTATTCGAAGGATTTCACCTGTTT |
| pTet_AK | pTet_For_A | ATGAAGACGTGGAGTCCCTATCAGTGATAGAGATTGAC | pTet_Rev_K | ACGAAGACCTGCATTTCGGTCAGTGCGTCCTGCTGATG |
| pTet_KB | pTet_For_K | ATGAAGACGTATGCTCCCTATCAGTGATAGAGATTGAC | pTet_Rev_B | ACGAAGACCTAGTATTCGGTCAGTGCGTCCTGCTGATG |
| pBad_AK | pBad_For_A | ATGAAGACGTGGAGAAGAAACCAATTGTCCATATTGCA | pBad_Rev_K | ACGAAGACCTGCATTATGGAGAAACAGTAGAGAGTTGC |
| pBad_KB | pBad_For_K | ATGAAGACGTATGCAAGAAACCAATTGTCCATATTGCA | pBad_Rev_B | ACGAAGACCTAGTATATGGAGAAACAGTAGAGAGTTGC |
| pSrpR_KB | pSrpR_For_K | ATGAAGACGTATGCTTCGTTACCAATTGACAGCTAGCT | pSrpR_Rev_B | ACGAAGACCTAGTAGTTTACAAACAAACAAGCATGTAT |
| pLmrA_FK | pLmrA_For_F | ATGAAGACGTCGCTTTCGTTACCAATTGACAACTGGTG | pLmrA_Rev_K | ACGAAGACCTGCATAAATATAGTGACTGGTCTATTATC |
| pBetI_EB | pBet_For_E | ATGAAGACGTGCTTTTCATGGATTCGTTACCAATTGAC | pBetI_Rev_B | ACGAAGACCTAGTAGCTAGCATTATATTGAACGTCCAA |
| pPhlF_GB | pPhlF_For_G | ATGAAGACGTTGCCTTCGTTACCAATTGACATGATACG | pPhlF_Rev_B | ACGAAGACCTAGTAACCTTAACGATACGGTACGTTTCG |
| pBM3R1_FB | pBM3R1_For_F | ATGAAGACGTCGCTTTCGTTACCAATTGACGGAATGAA | pBM3R1_Rev_B | ACGAAGACCTAGTAGCTAGCATTATCGGAATGAACGTT |
Primer Design for Fusion Proteins
| Device Name | Primer | Sequence |
|---|---|---|
| C0080_CI | C0080_Rev_I | ACGAAGACCTTAGACAACTTGACGGCTACATCATTCAC |
| C0040_CI | C0040_Rev_I | ACGAAGACCTTAGACAACTTGACGGCTACATCATTCAC |
| E0040m_ID | E0040m_For_I | ATGAAGACGTTCTAGAATGCGTAAAGGAGAAGAACTTTTC |
| E0030_ID | E0030_For_I | ATGAAGACGTTCTAGAATGGTGAGCAAGGGCGAGGAGCTG |
References
[1] A. Tamsir, J. Tabor, C. Voigt (2011). “Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’.” Nature 469: 212-215
