Difference between revisions of "Part:BBa K1041002:Experience"
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===Team NRP-UEA_Norwich 2013===
 
===Team NRP-UEA_Norwich 2013===
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Team NRP-UEA_Norwich 2013 created this part using biobricks [[BBa_K1041000]] and [[BBa_K1041001]]. These biobricks both contain a Nde1 site after their promoter sequence, enabling a restriction digest to be performed. The RFP coding gene was excised from BBa_K1041000 and ligated in front of the AntG promoter of BBa_K1041001 to create a new biobrick.
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Team NRP-UEA_Norwich 2013 created this part using biobricks [[BBa_K1041000]] and [[BBa_K1041001]]. These biobricks both contain an Nde1 site after their promoter sequence, enabling a restriction digest to be performed. The RFP coding gene was excised from BBa_K1041000 and ligated downstream of the AntG promoter of BBa_K1041001 to create this new biobrick.
  
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Due to the major research side of our project involving actinomycetes not ''E.coli'' this part was cloned into an appropriate actinomycete-specific intergrative plasmid PMS82. This was to allow the reporter to be utilised in screening for antimycin producing actinomycetes.
+
Due to the major research side of our project involving actinomycetes and not ''E. coli'' this part was also cloned into an appropriate actinomycete-specific integrative plasmid PMS82. This was to allow the reporter to be utilised in screening for antimycin producing actinomycetes. The plasmid containing the reporter sequence from [[Bba_K1041002]] was transformed into specialised ''E. coli''strains: ET12567 and pUZ8002. These cells were then conjugated with spore stocks of actinomycetes cultivated from soil samples to transfer the active biosensor in the plasmid.   
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The plasmid containing the reporter sequence (Bba_K1041002)was transformed into specialised ''E.coli''strains: ET12567 and pUZ8002. These cells were then conjugated with spore stocks of actinomycetes cultivated from soil samples to transfer the active biosensor in the plasmid.   
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One further experiment was designed to activate the RFP gene by transforming BBa_K1041002 into a BL21 ''E.Coli'' strain along with the plasmid pET28AntA which synthesises the sigma factor AntA. It was hoped that with both plasmids in the same cell, the AntA sigma factor would be produced and activate transcription of the RFP gene by binding to the promoter sequence antGP. This would mean that the cells could be spread onto agar plates containing Chlorophenicol and the colonies would appear red under natural light. Unfortunately the colonies did not appear red.  
+
One further experiment was designed to activate the RFP gene by transforming BBa_K1041002 into an ''E. coli'' BL21 strain along with the plasmid pET28AntA which synthesises the sigma factor AntA upon induction of T7 RNA polymerase by addition of IPTG. It was hoped that with both plasmids in the same cell, the AntA sigma factor would be produced and activate transcription of the RFP gene by binding to the promoter sequence antGP. This would mean that the cells could be spread onto agar plates containing Chloramphenicol and the colonies would appear red under natural light. The experiment was performed, but unfortunately the colonies did not appear red. To check that both plasmids were in the ''E. coli'' BL21 cells, DNA was purified from liquid cultures and analysed by restriction enzyme digests and agarose gel electrophoresis (''Fig 1''). The figure shows that both plasmids were contained within the ''E. coli'' BL21 cells as expected. Thus, further experiments are required to optimise this system and to discover why the sigma factor is not activating synthesis of the red fluorescence protein.  
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We then decided to confirm that both plasmids were in the BL21 cells by analysing the DNA by restriction enzyme digests and performing agarose gel electrophoresis, ''Fig 1''. The figure shows that both plasmids are contained within the BL21 cells as aspected and further experiments need to carried out to discover why the sigma factor is not activating transcription of the red fluorescence protein.  
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[[image:Bl21 Anta + Rfp digest.jpg|thumb|left|Fig 1: Analysis of 'E. coli'' BL21 cells containing pETAntA and BBa_K1041002 plasmids compared to the DNA of the plasmids alone digested with XbaI, NdeI and no enzyme.]]
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[[image:Bl21 Anta + Rfp digest.jpg|thumb|left|Fig 1:Analysis of BL21 cells containing pETAntA and BBa_K1041002 plasmids compared to the DNA of the plasmids alone digested with XbaI, NdeI and no enzyme.]]
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This involved sequencing, restriction digests and BLAST analysis.
 
This involved sequencing, restriction digests and BLAST analysis.
 
====Restriction Digest====
 
====Restriction Digest====
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Part Bba_K1041002 was digested with enzymes XbaI and NdeI and compared to uncut DNA ''Fig 2.''The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.  
+
Part Bba_K1041002 was digested with enzymes XbaI and NdeI and compared to uncut DNA (''Fig 2''). The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.  
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[[image:BIORAD 2013-09-18 16hr 02min 2.JPG|thumb|left|Fig 2:Lane 1 contains and Bba_K1041002 cut with XbaI and NdeI and lane 2 uncut Bba_K1041002.]]
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[[image:BIORAD 2013-09-18 16hr 02min 2.JPG|thumb|left|Fig 2: Lane 1 contains and Bba_K1041002 cut with XbaI and NdeI and lane 2 uncut Bba_K1041002.]]
  
 
<br>
 
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===Sequencing===
 
===Sequencing===
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The biobrick was sent off to a company for sequencing and the data we received back ''Fig 3,4,5'' showed the DNA is good quality as strong peaks were produced throughout analysis of the sample.   
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The biobrick was sent off to a company for sequencing and the data we received back (''Figs 3,4,5'') showed the DNA was good quality as strong chromatographic peaks were produced throughout analysis of the sample.   
  
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[[image:K1041002 1.JPG|thumb|left|Fig 3:K1041002 sequencing data part 1]]
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[[image:K1041002 1.JPG|thumb|left|Fig 3: K1041002 sequencing data part 1]]
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[[image:K1041002 2.JPG|thumb|left|Fig 4:K1041002 sequencing data part 2]]
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[[image:K1041002 2.JPG|thumb|left|Fig 4: K1041002 sequencing data part 2]]
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[[image:K1041002 3.JPG|thumb|left|Fig 5:K1041002 sequencing data part 3]]
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[[image:K1041002 3.JPG|thumb|left|Fig 5: K1041002 sequencing data part 3]]
  
 
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===BLAST Analysis===
 
===BLAST Analysis===
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The data we recieved back from the sequencing company was aligned using BLAST with the expected DNA sequence ''Fig 6,7''The sequencing with both the forward and reverse primers had over 96% matches.
+
The data we received back from the sequencing company was aligned using BLAST with the expected DNA sequence (''Figs 6,7''). The sequencing with both the forward and reverse primers had over 96% matches to the expected sequence. Deviations from the expected sequence were at the end of the useful sequencing data and combination of both sets of data confirmed a complete match with the expected sequence.
 
[[image:6 Fwd.JPG|thumb|left|Fig 6: Forward primer K1041002 sequencing data aligned with the expected DNA sequence]]
 
[[image:6 Fwd.JPG|thumb|left|Fig 6: Forward primer K1041002 sequencing data aligned with the expected DNA sequence]]
 
[[image:6 Rev.JPG|thumb|left|Fig 7: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence]]
 
[[image:6 Rev.JPG|thumb|left|Fig 7: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence]]

Revision as of 22:24, 4 October 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Team NRP-UEA_Norwich 2013

Team NRP-UEA_Norwich 2013 created this part using biobricks BBa_K1041000 and BBa_K1041001. These biobricks both contain an Nde1 site after their promoter sequence, enabling a restriction digest to be performed. The RFP coding gene was excised from BBa_K1041000 and ligated downstream of the AntG promoter of BBa_K1041001 to create this new biobrick.

Due to the major research side of our project involving actinomycetes and not E. coli this part was also cloned into an appropriate actinomycete-specific integrative plasmid PMS82. This was to allow the reporter to be utilised in screening for antimycin producing actinomycetes. The plasmid containing the reporter sequence from Bba_K1041002 was transformed into specialised E. colistrains: ET12567 and pUZ8002. These cells were then conjugated with spore stocks of actinomycetes cultivated from soil samples to transfer the active biosensor in the plasmid.

One further experiment was designed to activate the RFP gene by transforming BBa_K1041002 into an E. coli BL21 strain along with the plasmid pET28AntA which synthesises the sigma factor AntA upon induction of T7 RNA polymerase by addition of IPTG. It was hoped that with both plasmids in the same cell, the AntA sigma factor would be produced and activate transcription of the RFP gene by binding to the promoter sequence antGP. This would mean that the cells could be spread onto agar plates containing Chloramphenicol and the colonies would appear red under natural light. The experiment was performed, but unfortunately the colonies did not appear red. To check that both plasmids were in the E. coli BL21 cells, DNA was purified from liquid cultures and analysed by restriction enzyme digests and agarose gel electrophoresis (Fig 1). The figure shows that both plasmids were contained within the E. coli BL21 cells as expected. Thus, further experiments are required to optimise this system and to discover why the sigma factor is not activating synthesis of the red fluorescence protein.

Fig 1: Analysis of 'E. coli BL21 cells containing pETAntA and BBa_K1041002 plasmids compared to the DNA of the plasmids alone digested with XbaI, NdeI and no enzyme.



















Characterisation

This involved sequencing, restriction digests and BLAST analysis.

Restriction Digest

Part Bba_K1041002 was digested with enzymes XbaI and NdeI and compared to uncut DNA (Fig 2). The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.

Fig 2: Lane 1 contains and Bba_K1041002 cut with XbaI and NdeI and lane 2 uncut Bba_K1041002.



















Sequencing

The biobrick was sent off to a company for sequencing and the data we received back (Figs 3,4,5) showed the DNA was good quality as strong chromatographic peaks were produced throughout analysis of the sample.

Fig 3: K1041002 sequencing data part 1
Fig 4: K1041002 sequencing data part 2
Fig 5: K1041002 sequencing data part 3










BLAST Analysis

The data we received back from the sequencing company was aligned using BLAST with the expected DNA sequence (Figs 6,7). The sequencing with both the forward and reverse primers had over 96% matches to the expected sequence. Deviations from the expected sequence were at the end of the useful sequencing data and combination of both sets of data confirmed a complete match with the expected sequence.

Fig 6: Forward primer K1041002 sequencing data aligned with the expected DNA sequence
Fig 7: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence

User Reviews

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