Composite

Part:BBa_K415151:Experience

Designed by: Shirley Galbiati   Group: iGEM10_MIT   (2010-10-24)
Revision as of 15:08, 2 August 2011 by Allancrossman (Talk | contribs) (→‎User Reviews)

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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.

User Reviews

BBa_K415151 Allan Crossman (Edinburgh 2011)

The official sequencing verification from the Registry indicates that the BioBrick is not present in the plasmid that was sequenced, but instead has been replaced with part of the main E. coli genome sequence. If you direct your attention to [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=989379&to=991367&report=gbwithparts GenBank U00096.2 nucleotides 989379 - 991367] you will find a sequence with PstI and EcoRI sites at the end. This genomic DNA has evidently been cut with these enzymes and inserted into pSB1C3. This explains the Registry's sequencing results.

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UNIQ2d2f8ed7b542b0c7-partinfo-00000001-QINU UNIQ2d2f8ed7b542b0c7-partinfo-00000002-QINU