CCAUC tRNA (9-bp Anticodon) with RFP Reporter
From the BioBrick Parts Registry, and mutation of the RFP reporter, E1010, to add the 5-bp insertion.
Suppressor tRNA expressed by the medium-weak promoter, pBad, upstream of RFP with a 5-bp insertion, CCAUC. The tRNA is designed to suppress the insertion, allowing the appropriate translation of functional RFP.
See the following parts for more information: K199007, K199031 and K199009.
The template of the gene comes from BioBrick Part E1010. We designed a forward primer that included ATG, the 5-bp addition CCATC and the first 20 nucleotides on the 5' end of RFP after ATG. This made the edited 5' sequence for our RFP.
This RFP reporter gene has a 5-bp addition, CCAUC, after the start codon and before the rest of the gene. This is used in conjunction with the CCAUC tRNA (Parts K199007 and K199008). These CCAUC tRNAs suppress the frameshift caused by this 5-bp addition in the reporter.
We positioned the start codon, ATG, before the 5-bp codon and continued with the rest of the RFP gene. This way, the RBS wouldn't translate the reporter gene without reading over the 5-base codon first. In order to ligate our edited 5' sequence to the unchanged downstream portion of the RFP gene, we utilized the restriction enzyme NcoI that cuts at a single site 419-bp into BBa_E1010.
pBad-CCAUC Suppressor tRNA (9-bp Anticodon)
The pBad promoter is from the BioBrick Parts registry. Suppressor tRNA was made by de novo synthesis from single-stranded oligos. Based on the paper by Anderson et al. 2002.
Causes low expression of a suppressor tRNA that suppresses a 5-bp codon CCAUC, Parts K199007 and K199008.
We used the pBad promoter to transcribe lower levels of tRNA in a high copy plasmid, pSB1A2.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
CCAUC Suppressor tRNA (9-bp Anticodon and Produces Serine)
De Novo synthesis from oligos. Based on the paper by Anderson et al. (http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf)
This gene encodes for a 5 base pair tRNA suppressor that suppresses CCAUC, which would normally cause a frame shift mutation.
We used the 9 base anticodon loop that is complementary to the 5 base pair codon CCAUC.
Conversion of the iGEM parts registry to SBOL2.1