Difference between revisions of "Part:BBa K415151:Experience"

Latest revision as of 20:34, 2 August 2011

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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.

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.

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 verification sequencing 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 ends. This sequence (or rather, its reverse complement) perfectly matches both forward and reverse K415151 verification sequences. This genomic DNA has evidently been cut with these enzymes and has somehow become inserted into pSB1C3 via its EcoRI and PstI sites. This explains the Registry's sequencing results, as well as the lack of a proper prefix and suffix (since insertion via EcoRI and PstI deletes most of the prefix and most of the suffix).

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UNIQadf4ebd1ced53d9d-partinfo-00000001-QINU UNIQadf4ebd1ced53d9d-partinfo-00000002-QINU

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 <I>Allan Crossman (Edinburgh 2011)</I>
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-The [https://parts.igem.org/cgi/sequencing/one_blast.cgi?id=10599 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 may prefer to view the reverse complement) you will find a sequence with PstI and EcoRI sites at the ends. This genomic DNA has evidently been cut with these enzymes and has somehow become inserted into pSB1C3 via its EcoRI and PstI sites. This explains the Registry's sequencing results, as well as the lack of a complete prefix and suffix.
+The [https://parts.igem.org/cgi/sequencing/one_blast.cgi?id=10599 official verification sequencing] 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 ends. This sequence (or rather, its reverse complement) perfectly matches both forward and reverse K415151 verification sequences. This genomic DNA has evidently been cut with these enzymes and has somehow become inserted into pSB1C3 via its EcoRI and PstI sites. This explains the Registry's sequencing results, as well as the lack of a proper prefix and suffix (since insertion via EcoRI and PstI deletes most of the prefix and most of the suffix).
 |};