Difference between revisions of "Part:BBa K415151"

 
 
(One intermediate revision by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K415151 short</partinfo>
 
<partinfo>BBa_K415151 short</partinfo>
Line 7: Line 6:
 
The p8-GR1 part consists of an RBS, the p8 leader sequence, the zipper coil, an HA tag, a linker sequence, followed by a phage display optimized p8.  
 
The p8-GR1 part consists of an RBS, the p8 leader sequence, the zipper coil, an HA tag, a linker sequence, followed by a phage display optimized p8.  
  
−
<!-- Add more about the biology of this part here
+
 
−
===Usage and Biology===
+
  
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K415151 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K415151 SequenceAndFeatures</partinfo>
 +
<br><br>
 +
===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.
 +
 +
[[Image: Hyp_gr1_9_16_amp.jpg]]
 +
 +
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. The number of plaques for each dilution were counted.
 +
 +
 +
'''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 that when induced with AHL, cells transformed with K415151 and Hyperphage do not produce viable phage. 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]]
  
 +
The purple band at 13 kD represents the p8-GR1 fusion, and is evidence of the fusion protein's incorporation into the phage coat.
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K415151 parameters</partinfo>
 
<partinfo>BBa_K415151 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 00:50, 4 November 2010

K415010 + RBS : p8-GR1

This part is a composite part of BBa_K415010 (PluxR/cI : RBS : mCherry : Term : PtetR : RBS : LuxR:Term : Plux/cI) with a p8-GR1 fusion appended, where p8 is the major coat protein of M13 bacteriophage and GR1 is a leucine zipper coil that can bind to corresponding zipper coil, GR2. This part is designed to produce the fusion, which can then be displayed on the phage coat when used in a helper phage system.

The p8-GR1 part consists of an RBS, the p8 leader sequence, the zipper coil, an HA tag, a linker sequence, followed by a phage display optimized p8.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2368
  • 1000
    COMPATIBLE WITH RFC[1000]



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.

Hyp gr1 9 16 amp.jpg

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. The number of plaques for each dilution were counted.


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 that when induced with AHL, cells transformed with K415151 and Hyperphage do not produce viable phage. 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.

1023westernGR1.jpg

The purple band at 13 kD represents the p8-GR1 fusion, and is evidence of the fusion protein's incorporation into the phage coat.