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| + | Transformation of <i>luxCDABE</i> into pSB1AK3 |
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| - | WIKI CODING HERE
| + | * Attempted to transformed <i>luxCDABE</i> into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight. |
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| - | CHINMOYEE
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| + | Digest Verification |
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| - | | + | *Performed a verification digest with NotI of BBK LuxOD47E in psB1AC3- colonies 3/4 cut with EcoRI/PstI and colonies 2/3 cut with XbaI/PstI. This is one last verification that our gene of interest (LuxOD47E) is in the psB1AC3 vector. If successful, cut lanes should contain 2 bands at ~1.4 for the gene and ~3 kb for the vector, Uncut lanes should only contain one band at ~4 kb, representing the vector with the gene of interest inside. |
| - | *Protocol: Used GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH2O. | + | *Ran products on a 1% agarose gel with 1.0kb+ GeneRuler DNA Ladder. |
| - | <Br>
| + | *Ran each cut colony with a uncut colony as a control. |
| - | | + | *See gel photo below. Lanes 1,3,5 and 7 are cut DNA- EcoRI/PstI colonies 2 and 4 and XbaI/PstI colonies 2 and 3 respectively, lanes 2,4,6 and 8 are all uncut colonies. |
| - | *Results
| + | *Analysis: Colonies cut with EcoRI/PstI have bands much higher than expected (~1.4 kb and ~3.0 kb in the cut lanes). XbaI/PstI-cut colony 3 does not show two bands in the cut lane and the bands are not the expected size. XbaI/PstI-cut colony 2 does show two bands in the cut lane at around the expected sizes and the uncut lane has only 1 band at around 4 kb, as expected. We will proceed with this colony, sending it down for sequencing. |
| - | | + | *Sent down 10uL XbaI-cut Colony 2 with BBK_CP_F and BBK_CP_R primers, results should be in by Monday. |
| - | {| border="1"
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| - | | '''Plasmid''' || '''260/280''' || '''260/230''' || '''Concentration [ng/μL]'''
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| - | | ''psB1AC3 1 || 1.96 || 3.48 || 76.9
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| - | | ''psB1AC3 2 || 1.93 || 2.29 || 98.1
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| - | | ''psB1AC3 3 || 1.87|| 1.88|| 48.5
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| - | | ''psB1AC3 4|| 1.90|| 2.11|| 118.9
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| - | | ''psB1AK3 1 || 2.05 || 2.42 || 134.4
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| - | | ''psB1AK3 2 || 1.95 || 2.23 || 55.6
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| - | | ''psB1AK3 3 || 1.91 || 2.22 || 97.9
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| - | | ''psB1AK3 4 || 2.03 || 1.92 || 44.5
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| - | Verification digest to verify the presence of restriction sites in the psB1AC3, psB1AK3 vectors as well as in our TOPO TA vector with LuxOD47E.
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| - | *Protocol: | + | |
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| - | 5 reactions for psB1AC3 vector, 5 reactions for psB1AK3 vector, 1 reaction for gene of interest (LuxOD47E in pCR2.1-TOPO vector).
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| - | BBK Vector Reaction List
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| - | Reaction 1- Not1 + React 3 Buffer
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| - | Reaction 2- EcoRI + SpeI + React Buffer 4
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| - | Reaction 3- XbaI + PstI + React 2 Buffer
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| - | Reaction 4- EcoRI + PstI + React 2 Buffer
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| - | Reaction 5- XbaI + SpeI + React 4 Buffer
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| - | BBK vector tube preparation
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| - | 8 uL Plasmid (psB1AC3 or psB1AK3)
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| - | 2 uL appropriate React Buffer | + | |
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| - | 1 uL of each appropriate restrcition enzyme | + | |
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| - | ddH20 up to 20 uL
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| - | TOPO vector tube preparation
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| - | 10 uL DNA (LuxOD47E in pCR2.1- TOPO vector)
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| - | 7 uL ddH20
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| - | 2 uL React 3 Buffer | + | |
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| - | 1 uL NotI restriction enzymes
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| - | Left to digest overnight in waterbath at 37°C.
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| + | Phosphatase, Ligation and Transformation of LuxPQ into BBK vector |
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| - | WIKI CODING HERE
| + | Purpose: to construct and transform the LuxPQ insert and psB1AC3 insert in order to standardize the genetic part for future construction. The products from the overnight digest (July 1) underwent phosphatase treatment and subsequent ligation and transformation into TOP10 competent cells. The cells were then plated on LB+chlor plates. |
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| - | WIKI CODING HERE
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| | For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue. | | For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue. |
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| + | Research in Antifreeze Activity |
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| - | WIKI CODING HERE
| + | We want to look at AFP, which has a very different structure in different organisms. One that we would be able to work with best (or more efficiently with) would be from gram negative bacteria. So I looked at <i>Pseudomonas Putida</i>, <i>Micrococcus cryophilus</i>, <i>Rhodococcus erythropolis</i>, and <i>Marinomonas protea primoryensis</i>. Some are psychrophilic and halophilic, so those might not be ideal for us. |
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| - | WIKI CODING HERE
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| - | WIKI CODING HERE
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| | The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls. | | The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls. |
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| | These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today. | | These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today. |
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CAROL
Transformation of luxCDABE into pSB1AK3
- Attempted to transformed luxCDABE into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight.
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EMILY
Digest Verification
- Performed a verification digest with NotI of BBK LuxOD47E in psB1AC3- colonies 3/4 cut with EcoRI/PstI and colonies 2/3 cut with XbaI/PstI. This is one last verification that our gene of interest (LuxOD47E) is in the psB1AC3 vector. If successful, cut lanes should contain 2 bands at ~1.4 for the gene and ~3 kb for the vector, Uncut lanes should only contain one band at ~4 kb, representing the vector with the gene of interest inside.
- Ran products on a 1% agarose gel with 1.0kb+ GeneRuler DNA Ladder.
- Ran each cut colony with a uncut colony as a control.
- See gel photo below. Lanes 1,3,5 and 7 are cut DNA- EcoRI/PstI colonies 2 and 4 and XbaI/PstI colonies 2 and 3 respectively, lanes 2,4,6 and 8 are all uncut colonies.
- Analysis: Colonies cut with EcoRI/PstI have bands much higher than expected (~1.4 kb and ~3.0 kb in the cut lanes). XbaI/PstI-cut colony 3 does not show two bands in the cut lane and the bands are not the expected size. XbaI/PstI-cut colony 2 does show two bands in the cut lane at around the expected sizes and the uncut lane has only 1 band at around 4 kb, as expected. We will proceed with this colony, sending it down for sequencing.
- Sent down 10uL XbaI-cut Colony 2 with BBK_CP_F and BBK_CP_R primers, results should be in by Monday.
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JEREMY
Phosphatase, Ligation and Transformation of LuxPQ into BBK vector
Purpose: to construct and transform the LuxPQ insert and psB1AC3 insert in order to standardize the genetic part for future construction. The products from the overnight digest (July 1) underwent phosphatase treatment and subsequent ligation and transformation into TOP10 competent cells. The cells were then plated on LB+chlor plates.
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KEVIN
Continuation of the research in Antifreeze proteins
Usefulness of the signalling system in antifreeze activity
For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue.
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MANDY
Research in Antifreeze Activity
We want to look at AFP, which has a very different structure in different organisms. One that we would be able to work with best (or more efficiently with) would be from gram negative bacteria. So I looked at Pseudomonas Putida, Micrococcus cryophilus, Rhodococcus erythropolis, and Marinomonas protea primoryensis. Some are psychrophilic and halophilic, so those might not be ideal for us.
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VICKI
Repeat of colony PCR of J13002 + LuxOD47A + B0015
The purpose and materials/methods are the same as they were yesterday. This time, only 4 colonies were tested, along with the same size controls as yesterday.
Results:
The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.
Plasmid isolation and NotI restriction digest
Purpose: To further verify if the J13002 + LuxOD47A + B0015 was successfully biobricked into the colonies.
Materials and methods:
These were done in accordance with the procedure outlined on the protocol page.
Results:
These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.
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