Difference between revisions of "Part:BBa K415151:Experience"
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'''Assessing p8-GR1 incorporation into phage coat''' | '''Assessing p8-GR1 incorporation into phage coat''' | ||
| − | We transformed XL1-Blue cells with BBa_K415151, grew cells up without induction and infected with M13K07 helper phage. After 5 hours, the phage was purified, denatured, and western blotted with an anti-HA antibody. | + | MIT 2010 designed this part with the intent of displaying it on polyphage. When this part (on the pSB1A3 backbone) was co-transformed with hyperphage in DH5a-pro cells and induced with AHL, we observed no polyphage on the cells. |
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| + | [[Image: Hyp_gr1_9_16_amp.jpg]] | ||
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| + | We hypothesized that we were overexpressing the p-GR1, which was interfering with phage assembly. We decided to quantify the amount of phage that was being produced in samples that were induced with AHL and samples that were not induced. To do this, we co-transformed M13K07 and K415151, grew 3mL cultures for 3 hours, induced +/- AHL, and grew for an additional 7 hours. We took the supernatants, which contained the phage produced. The supernatants were titered to quantify the phage. | ||
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| + | |||
| + | '''Titer Results''' | ||
| + | |||
| + | {|| | ||
| + | {|style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000" | ||
| + | |- | ||
| + | !style="border-style: solid; border-width: 1px"| Sample | ||
| + | !style="border-style: solid; border-width: 1px"| AHL | ||
| + | !style="border-style: solid; border-width: 1px"| 10^0 | ||
| + | !style="border-style: solid; border-width: 1px"| 10^-2 | ||
| + | !style="border-style: solid; border-width: 1px"| 10^-4 | ||
| + | !style="border-style: solid; border-width: 1px"| 10^-6 | ||
| + | !style="border-style: solid; border-width: 1px"| 10^-8 | ||
| + | |- | ||
| + | |style="border-style: solid; border-width: 1px"| M13KO7+151 | ||
| + | |style="border-style: solid; border-width: 1px"| + | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |- | ||
| + | |style="border-style: solid; border-width: 1px"| M13KO7+151 | ||
| + | |style="border-style: solid; border-width: 1px"| - | ||
| + | |style="border-style: solid; border-width: 1px"| inc | ||
| + | |style="border-style: solid; border-width: 1px"| p | ||
| + | |style="border-style: solid; border-width: 1px"| 100 | ||
| + | |style="border-style: solid; border-width: 1px"| 4 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |- | ||
| + | |style="border-style: solid; border-width: 1px"| M13KO7 | ||
| + | |style="border-style: solid; border-width: 1px"| - | ||
| + | |style="border-style: solid; border-width: 1px"| na | ||
| + | |style="border-style: solid; border-width: 1px"| 5 | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |style="border-style: solid; border-width: 1px"| inc | ||
| + | |style="border-style: solid; border-width: 1px"| 0 | ||
| + | |- | ||
| + | |style="border-style: solid; border-width: 1px"| M13KE3 Stock | ||
| + | |style="border-style: solid; border-width: 1px"| - | ||
| + | |style="border-style: solid; border-width: 1px"| na | ||
| + | |style="border-style: solid; border-width: 1px"| na | ||
| + | |style="border-style: solid; border-width: 1px"| na | ||
| + | |style="border-style: solid; border-width: 1px"| n | ||
| + | |style="border-style: solid; border-width: 1px"| 100 | ||
| + | |} | ||
| + | | | ||
| + | Key: | ||
| + | * na = not attempted | ||
| + | * inc = inconclusive (mishandling of plates) | ||
| + | * p = mostly plaque or uncountable number of plaques | ||
| + | * n = no growth | ||
| + | |} | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | The M13KO7 results are strange, since in previous titers we have obtained 652 plaques on the 10^4 dilution for M13KO7, so we cannot conclusively compare the infectivity to M13KO7. M13KE3 is a different strain of M13. We used a purified stock as a positive control. However, we do have an indication of AHL dependence for the viability of the phage when M13KO7 is co-transformed with K415151. In our subsequent experiments, we chose not to induce with AHL in order to guarantee phage production. | ||
| + | |||
| + | Our next question was whether the basal expression of the K415151 part was enough for p8-GR1 to be incorporated into the phage coat. We transformed XL1-Blue cells with BBa_K415151, grew cells up without induction and infected with M13K07 helper phage. After 5 hours, the phage was purified, denatured, and western blotted with an anti-HA antibody. We found that the p8-GR1 was incorporated into the phage coat. | ||
[[Image:1023westernGR1.jpg]] | [[Image:1023westernGR1.jpg]] | ||
Revision as of 21:46, 28 October 2010
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how you used this part and how it worked out.
Applications of BBa_K415151
Assessing p8-GR1 incorporation into phage coat
MIT 2010 designed this part with the intent of displaying it on polyphage. When this part (on the pSB1A3 backbone) was co-transformed with hyperphage in DH5a-pro cells and induced with AHL, we observed no polyphage on the cells.
We hypothesized that we were overexpressing the p-GR1, which was interfering with phage assembly. We decided to quantify the amount of phage that was being produced in samples that were induced with AHL and samples that were not induced. To do this, we co-transformed M13K07 and K415151, grew 3mL cultures for 3 hours, induced +/- AHL, and grew for an additional 7 hours. We took the supernatants, which contained the phage produced. The supernatants were titered to quantify the phage.
Titer Results
| Sample | AHL | 10^0 | 10^-2 | 10^-4 | 10^-6 | 10^-8 |
|---|---|---|---|---|---|---|
| M13KO7+151 | + | 0 | 0 | 0 | 0 | 0 |
| M13KO7+151 | - | inc | p | 100 | 4 | 0 |
| M13KO7 | - | na | 5 | 0 | inc | 0 |
| M13KE3 Stock | - | na | na | na | n | 100 |
Key:
- na = not attempted
- inc = inconclusive (mishandling of plates)
- p = mostly plaque or uncountable number of plaques
- n = no growth
The M13KO7 results are strange, since in previous titers we have obtained 652 plaques on the 10^4 dilution for M13KO7, so we cannot conclusively compare the infectivity to M13KO7. M13KE3 is a different strain of M13. We used a purified stock as a positive control. However, we do have an indication of AHL dependence for the viability of the phage when M13KO7 is co-transformed with K415151. In our subsequent experiments, we chose not to induce with AHL in order to guarantee phage production.
Our next question was whether the basal expression of the K415151 part was enough for p8-GR1 to be incorporated into the phage coat. We transformed XL1-Blue cells with BBa_K415151, grew cells up without induction and infected with M13K07 helper phage. After 5 hours, the phage was purified, denatured, and western blotted with an anti-HA antibody. We found that the p8-GR1 was incorporated into the phage coat.
The purple band at 13 kD represents the p8-GR1 fusion, and is evidence of the fusion protein's incorporation into the phage coat.
User Reviews
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