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| | * Overnight plates resulted in no colonies | | * Overnight plates resulted in no colonies |
| | * Attempted other types of ligase (Invitrogen T4 overnight at 16 degrees Celsius and NEB T4 Ligase 1 hour at room temperature) and products were transformed into Top10 cells. The cells were plated on AK/AC plates. | | * Attempted other types of ligase (Invitrogen T4 overnight at 16 degrees Celsius and NEB T4 Ligase 1 hour at room temperature) and products were transformed into Top10 cells. The cells were plated on AK/AC plates. |
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| | Looked into Papers : | | Looked into Papers : |
| - | <br>Parameter estimation in stochastic biochemical reactions
| + | *Parameter estimation in stochastic biochemical reactions |
| - | <br>Parameter estimation and determinability analysis applied to Drosophila gap gene circuits
| + | *Parameter estimation and determinability analysis applied to Drosophila gap gene circuits |
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| | *Did a plasmid Isolation of Colony2X LuxOD47E BBK. | | *Did a plasmid Isolation of Colony2X LuxOD47E BBK. |
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| - | *Today I did a colony PCR with p Taq and Biobrick primers (BBK-CP-F and BBK-CP-R) to see if we were able to clone in either the J13002 promoter or the B0015 terminator to our gene of interest, LuxOD47E. PCR products were run on a 1% agarose gel with LuxOD47E BBK as a size control and ddH2O as a negatve control. Gel if pictures below. Lanes 1-4 are J13002-LuxOD47E and Lanes 5-8 are LuxOD47E-B0015. Lane 9 is LuxOD47E BBK, lanes 10 and 11 are left blank and Lane 12 is a negative control with ddH2O. | + | *Today I did a colony PCR with p Taq and Biobrick primers (BBK-CP-F and BBK-CP-R) to see if we were able to clone in either the J13002 promoter or the B0015 terminator to our gene of interest, BBK LuxOD47E. PCR products were run on a 1% agarose gel with LuxOD47E BBK as a size control and ddH2O as a negative control. Gel if pictures below. Lanes 1-4 are J13002-LuxOD47E and Lanes 5-8 are LuxOD47E-B0015. Lane 9 is LuxOD47E BBK, lanes 10 and 11 are left blank and Lane 12 is a negative control with ddH2O. |
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| - | *From this gel it looks like the cloning of J13002 may have been successful as the band sin the first four lanes appear slighlty higher than the band in the size control lane. We will make overnight cultures of these colonies, isolate plasmid and send the colony with the best concentration for DNA sequencing.
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| - | | + | *From this gel it looks like the cloning of J13002 may have been successful as the band sin the first four lanes appear slightly higher than the band in the size control lane. We will make overnight cultures of these colonies, isolate plasmid and send the colony with the best concentration for DNA sequencing. |
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| | Purpose: To move LuxPQ from psB1AC3 into psB1AK3. Colonies 6 and 7 (see July 3 – verified presence of LuxPQ) of LuxPQ in AC and the AK vector were cut with the following combinations of enzymes: XbaI and PstI; EcoRI and PstI. After this restriction digest, phosphotase and ligation treatment was performed and the plasmids were subsequently transformed into TOP10 competent cells and plated overnight. | | Purpose: To move LuxPQ from psB1AC3 into psB1AK3. Colonies 6 and 7 (see July 3 – verified presence of LuxPQ) of LuxPQ in AC and the AK vector were cut with the following combinations of enzymes: XbaI and PstI; EcoRI and PstI. After this restriction digest, phosphotase and ligation treatment was performed and the plasmids were subsequently transformed into TOP10 competent cells and plated overnight. |
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| | The ligated construct of J13002 and GFP/RFP were transformed into TOP10 cells. This was done in order to test the functionality of the fluorescent proteins and the promoter. These have to grow overnight. | | The ligated construct of J13002 and GFP/RFP were transformed into TOP10 cells. This was done in order to test the functionality of the fluorescent proteins and the promoter. These have to grow overnight. |
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CAROL
Transformation
- Overnight plates resulted in no colonies
- Attempted other types of ligase (Invitrogen T4 overnight at 16 degrees Celsius and NEB T4 Ligase 1 hour at room temperature) and products were transformed into Top10 cells. The cells were plated on AK/AC plates.
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CHINMOYEE
Research : How to Optimize parameters for the model ?
Looked into Papers :
- Parameter estimation in stochastic biochemical reactions
- Parameter estimation and determinability analysis applied to Drosophila gap gene circuits
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EMILY
Colony PCR for Verification of Construction
- Did a plasmid Isolation of Colony2X LuxOD47E BBK.
- Today I did a colony PCR with p Taq and Biobrick primers (BBK-CP-F and BBK-CP-R) to see if we were able to clone in either the J13002 promoter or the B0015 terminator to our gene of interest, BBK LuxOD47E. PCR products were run on a 1% agarose gel with LuxOD47E BBK as a size control and ddH2O as a negative control. Gel if pictures below. Lanes 1-4 are J13002-LuxOD47E and Lanes 5-8 are LuxOD47E-B0015. Lane 9 is LuxOD47E BBK, lanes 10 and 11 are left blank and Lane 12 is a negative control with ddH2O.
- From this gel it looks like the cloning of J13002 may have been successful as the band sin the first four lanes appear slightly higher than the band in the size control lane. We will make overnight cultures of these colonies, isolate plasmid and send the colony with the best concentration for DNA sequencing.
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JAMIE
Plasmid construct of luxOU construct into psB1AK3
B0015-R0040-luxOU-B0015 is in psB1AC3. In order to allow for construction of signaling circuit from two approaches, a plasmid switch was done into psB1AK3. Verification involved colony PCR using luxOU construct in psB1AC3 as a positive control (July 8 2009). Plasmid miniprep was done and NotI verification digest was done (July 9 2009).
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JEREMY
Plasmid Switch of LuxPQ from AC to AK
Purpose: To move LuxPQ from psB1AC3 into psB1AK3. Colonies 6 and 7 (see July 3 – verified presence of LuxPQ) of LuxPQ in AC and the AK vector were cut with the following combinations of enzymes: XbaI and PstI; EcoRI and PstI. After this restriction digest, phosphotase and ligation treatment was performed and the plasmids were subsequently transformed into TOP10 competent cells and plated overnight.
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KEVIN
Transformation
The ligated construct of J13002 and GFP/RFP were transformed into TOP10 cells. This was done in order to test the functionality of the fluorescent proteins and the promoter. These have to grow overnight.
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PRIMA
Phosphatase Treatment
I shadowed Carol today. She let me do the phosphatase treatment.
Purpose: Adding phosphatase to her vector. (at this point i didn't know much about her project or what she was doing. I just focused on the techniques). Next step is to ligate the insert and the vector. Finally, we transformed the plasmid into top 10 cells and plated it.
I continued to follow up with the old companies. I mostly left voicemails to the company staff who were away from the desk. I sent our sponsorship packages out to companies who expressed interest in our project.
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