IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/09

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(Floor two)
Current revision (12:45, 30 September 2013) (view source)
(Floor One)
 
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==Floor One==
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Made up 4 l LB agar media for the spore titres - 10 g Difco Bacto tryptone, 5 g Difco yeast extract, 5 g, 5 g NaCl, 1 g glucose, 15 g agar, 1000 ml distilled water. Autoclaved and poured into plates.
 +
 +
More contaminated plates were streak-purified.
 +
 +
Single Streptomyces colonies were lawned.
 +
 +
Spore stocks were prepared, filtered and resuspended in glycerol (only as much as required to suspend).
 +
 +
We have arrived at 3 options for the Biosensor/reporter construct:
 +
- Neo construct utilised at various concentrations of kanamycin to attempt to produce AntA constitutively.
 +
- RFP reporter gene - only affects the structure of bacteria, not function (non-lethal), and can visualise the production of AntA. Could also be used to optimise E. coli expression.
 +
- GUS reporter gene (β-glucoronidase enzyme) - also a non-lethal type of selection (Myronovskyi ''et al'', 2011). Works like a blue-white screen where a substrate (glucoronate) is broken down to produce a blue ring/halo around a colony and will remain even if AntA ceases to be produced.
 +
All of these constructs would have to be made iGEM-compatible.
 +
We still need to carry out Bioassays to identify active anti-fungal metabolites (against Candida albicans) and AntA PCRS (with degenerative primers) to identify AntGp alongside screening which ultimately confirms the presence of AntA in the colonies. These methods are still much cheaper than current techniques that screen the entire genome, as it leads to only a small section of the genome being sequenced.
 +
It is still useful to have the whole genome sequence as well, for example, a cluster may exist that is not in the typical reported genome sequence for the strain.
 +
We will also run 16S PCR sequencing to find out what the Streptomyces strains are and spore titres of the spore stocks to estimate the number of spores present - bioassays require a certain number of spores and the strains we have are highly variable in spore number.
 +
 +
Performed dilutions of the soil samples up to 78 (10^-1 to 10^-4). Plated the dilutions 10^-2 to 10^-4 on SFM + Ny.
 +
 +
 +
Reference:
 +
Myronovskyi M., Welle E., Fedorenko V., Luzhetskyy A. (2011). Beta-glucuronidase as a sensitive and versatile reporter in actinomycetes. Applied and environmental microbiology 77, 5370–83.
 +
==Floor two==
==Floor two==
-
Another restriction digest of all the biobricks was prepared with different enzymes, to produce larger fragments. We used the enzymes PvuII and Xho1 to enable us to distinguish between the biobricks as the digests, when analysed using agarose gel electorphoresis,  should produce different size fragments for each plasmid.  
+
Another restriction digest of all the biobricks was prepared with different enzymes, to produce larger fragments. We used the enzymes PvuII and Xho1 to enable us to distinguish between the biobricks as the digests, when analysed using agarose gel electorphoresis,  produced different size fragments for each plasmid. A sample was also cut with EcoRI and PstI ''fig 2,3''. <br>
-
[[Image:BIORAD 2013-08-09 16hr 21min.jpg|thumb|Fig. Analysis of restriction digest of biobricks using enzymes PvuII and XhoI. Lanes 1,3,5,7,9 contain samples 3b2,1 colony 1,1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with PvuII. Lanes 2,4,6,8,10 contain samples 3b2, 1 colony 1, 1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with XhoI.]]
+
The plasmid Pet28AntA which produces the σ factor AntA was transformed into BL21 and BL21 star competent ''E.coli''cells. The four plates were left to incubate over the weekend at room temperature.
-
[[Image:BIORAD 2013-08-09 16hr 23min.jpg|thumb|Fig. Analysis of restriction digest of biobricks using enzymes PvuII, XhoI, EcoRI and PstI. Lanes 1,3,5,7, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with PvuII. Lanes 2,4,6,8, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with XhoI. Lanes 10,11,12 contain sample 3c cut with EcoRI, PstI and No enzyme respectively]]
+
[[Image:BIORAD 2013-08-09 16hr 21min.jpg|thumb|Fig 2. Analysis of restriction digest of biobricks using enzymes PvuII and XhoI. Lanes 1,3,5,7,9 contain samples 3b2,1 colony 1,1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with PvuII. Lanes 2,4,6,8,10 contain samples 3b2, 1 colony 1, 1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with XhoI.]]
 +
[[Image:BIORAD 2013-08-09 16hr 23min.jpg|thumb|Fig 3. Analysis of restriction digest of biobricks using enzymes PvuII, XhoI, EcoRI and PstI. Lanes 1,3,5,7, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with PvuII. Lanes 2,4,6,8, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with XhoI. Lanes 10,11,12 contain sample 3c cut with EcoRI, PstI and No enzyme respectively]]
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Floor One

Made up 4 l LB agar media for the spore titres - 10 g Difco Bacto tryptone, 5 g Difco yeast extract, 5 g, 5 g NaCl, 1 g glucose, 15 g agar, 1000 ml distilled water. Autoclaved and poured into plates.

More contaminated plates were streak-purified.

Single Streptomyces colonies were lawned.

Spore stocks were prepared, filtered and resuspended in glycerol (only as much as required to suspend).

We have arrived at 3 options for the Biosensor/reporter construct: - Neo construct utilised at various concentrations of kanamycin to attempt to produce AntA constitutively. - RFP reporter gene - only affects the structure of bacteria, not function (non-lethal), and can visualise the production of AntA. Could also be used to optimise E. coli expression. - GUS reporter gene (β-glucoronidase enzyme) - also a non-lethal type of selection (Myronovskyi et al, 2011). Works like a blue-white screen where a substrate (glucoronate) is broken down to produce a blue ring/halo around a colony and will remain even if AntA ceases to be produced. All of these constructs would have to be made iGEM-compatible. We still need to carry out Bioassays to identify active anti-fungal metabolites (against Candida albicans) and AntA PCRS (with degenerative primers) to identify AntGp alongside screening which ultimately confirms the presence of AntA in the colonies. These methods are still much cheaper than current techniques that screen the entire genome, as it leads to only a small section of the genome being sequenced. It is still useful to have the whole genome sequence as well, for example, a cluster may exist that is not in the typical reported genome sequence for the strain. We will also run 16S PCR sequencing to find out what the Streptomyces strains are and spore titres of the spore stocks to estimate the number of spores present - bioassays require a certain number of spores and the strains we have are highly variable in spore number.

Performed dilutions of the soil samples up to 78 (10^-1 to 10^-4). Plated the dilutions 10^-2 to 10^-4 on SFM + Ny.


Reference: Myronovskyi M., Welle E., Fedorenko V., Luzhetskyy A. (2011). Beta-glucuronidase as a sensitive and versatile reporter in actinomycetes. Applied and environmental microbiology 77, 5370–83.

Floor two

Another restriction digest of all the biobricks was prepared with different enzymes, to produce larger fragments. We used the enzymes PvuII and Xho1 to enable us to distinguish between the biobricks as the digests, when analysed using agarose gel electorphoresis, produced different size fragments for each plasmid. A sample was also cut with EcoRI and PstI fig 2,3.
The plasmid Pet28AntA which produces the σ factor AntA was transformed into BL21 and BL21 star competent E.colicells. The four plates were left to incubate over the weekend at room temperature.

Fig 2. Analysis of restriction digest of biobricks using enzymes PvuII and XhoI. Lanes 1,3,5,7,9 contain samples 3b2,1 colony 1,1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with PvuII. Lanes 2,4,6,8,10 contain samples 3b2, 1 colony 1, 1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with XhoI.
Fig 2. Analysis of restriction digest of biobricks using enzymes PvuII and XhoI. Lanes 1,3,5,7,9 contain samples 3b2,1 colony 1,1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with PvuII. Lanes 2,4,6,8,10 contain samples 3b2, 1 colony 1, 1 colony 2, 2 colony 1, 2 colony 2 respectively, which were cut with XhoI.
Fig 3. Analysis of restriction digest of biobricks using enzymes PvuII, XhoI, EcoRI and PstI. Lanes 1,3,5,7, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with PvuII. Lanes 2,4,6,8, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with XhoI. Lanes 10,11,12 contain sample 3c cut with EcoRI, PstI and No enzyme respectively
Fig 3. Analysis of restriction digest of biobricks using enzymes PvuII, XhoI, EcoRI and PstI. Lanes 1,3,5,7, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with PvuII. Lanes 2,4,6,8, contain samples 2b1,2b2,2c1,2c2 respectively, which were cut with XhoI. Lanes 10,11,12 contain sample 3c cut with EcoRI, PstI and No enzyme respectively


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