Parts from the Standard Registry of parts, M13K07, and my refactored genes.
This insert, when placed in between HpaI and BamHI restriction sites in M13K07, in the right orientation, should be capable of infecting E.coli and, when the environment has high molarity, the phage produced will express mCherry on their coat.
I had to consider the order in which to place everything and carefully refactor some of the basic parts I will use.
New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene X message (BBa_M13010). The part aligns with base pairs 480-495 in M13K07 genome. It was identified as the RBS for gene II message by Wezenbeek et al in Gene (1980) 11: 129-148
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene V message (BBa_M13005). The part aligns with base pairs 827-842 in M13K07 genome. It was identified as the RBS for the gene V message by Wezenbeek et al in Gene (1980) 11: 129-148
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene VII message (BBa_M13007). The part aligns with base pairs 1092-1107 in M13K07 genome. It was identified as the RBS for gene VII message by Wezenbeek et al in Gene (1980) 11: 129-148
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene IX message (BBa_M13009). The part aligns with base pairs 1190-1205 in M13K07 genome. It was identified as the RBS for gene IX message by Wezenbeek et al in Gene (1980) 11: 129-148
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene VIII message (BBa_M13008). The part aligns with base pairs 1285-1300 in M13K07 genome. It was identified as the RBS for gene VIII message by Wezenbeek et al in Gene (1980) 11: 129-148
New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban.
This part is from the bacteriophage M13 genome, bp 381-428 in M13K07. It directs transcription of M13 gene X (BBa_M13510, BBa_M13010). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
This part is from the bacteriophage M13 genome, bp 786-835 in M13K07. It directs transcription of M13 gene V (BBa_M13505, BBa_M13005). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
This part is from the bacteriophage M13 genome, bp 1155-1201 in M13K07. It directs transcription of M13 gene VIII (BBa_M13508, BBa_M13008). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
This part is from the bacteriophage M13 genome, bp 1500-1547 in M13K07. It directs transcription of M13 gene III (BBa_M13503, BBa_M13003). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
It is the second half of gene II after HpaI cuts gene II. I removed the Gene X Promoter and RBS sequences.
I altered the Gene X Promoter and RBS sequences
This part produces the GEne X protein but does not contain a promoter for Gene V.
I actually didn't have to remove the RBS for Gene V since it is in a space between Genes X and V. I only removed the Gene V promoter.
This produces Protein V and still has Gene VII promoter (if one exists) intact. It does not have a Gene VII RBS.
I could not destroy the Gene VII promoter even though this would be ideal for refactoring. This promoter has not been identified, but I suspect that, if it exists, it sits somewhere in this gene.
Gene VII w/o Genes IX RBS or VIII Promoter
This produces Protein VII but does not contain the promoter region for Genes VIII or RBS for Gene IX. It may contain a Gene IX promoter.
I realized that the Gene VIII promoter is actually within this gene, which surprised me, and I had to remove that along with the RBS for Gene IX. I suspect that the Gene IX promoter, should one exist, may be in this gene.
This produces Protein IX but does not contain the Gene VIII RBS.
This produces the Gene VIII protein but does not have the Gene III Promoter.
I had to remove the Gene III Promoter, but the Gene III RBS site is between Genes VIII and III, so I didn't have to worry about that.
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene III message (BBa_M13003). The part aligns with base pairs 1563-1578 in M13K07 genome. It was identified as the RBS for gene III message by Wezenbeek et al in Gene (1980) 11: 129-148 New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban
Gene VIII w/o Gene III Promoter + w/ mCherry
M13K07 and the Standard Registry of Parts, thanks to work done in Roger Tsien's laboratory at UCSD.
This produces gene VIII (without the Gene III promoter) with mCherry fused to it.
I had to remove the Gene III Promoter. Then I had to try to add the mCherry onto the extracellular side of protein VIII, which I believe is the N-terminus, which I think is coded on the 5' side of the gene, so I added mCherry before Gene VIII.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
This produces the first half of Protein III, up to where Gene III is cut by BamHI.
I had to find out where BamHI cuts this gene.
Conversion of the iGEM parts registry to SBOL2.1