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Questioning the Dogma of the Central Dogma Introduc)on
The flow of gene)c informa)on from DNA to RNA to protein is the commonly accepted dogma of molecular biology. Experimental results, however, are not always consistent with the central dogma. The goal of this project is to test the validity of several aspects of the central dogma, including bidirec)onal promoters, mul)ple start codons, and codon op)miza)on.
Bidirec)onal Promoters Mul)ple Start Codons Codon Op)miza)on
ATG [N]ATG NRFP sfGFP
Objec&ve • What happens if there are mul)ple start codons on a single mRNA
strand? • Which start codon is selected for the ini)a)on of protein synthesis?
Rela)ve flu
orescence
Fluorescence of Threonine 6-‐Repeat Codon Sequences
Objec&ve • How does codon bias affect protein transla)on rate? • What is the op)mized codon for threonine?
GFP mCherry
AHL leader sequence
6 codon repeat sequence
Objec&ve • What direc)on does a promoter promote in? • How biased is a promoter in each direc)on? • How do palindromic promoters affect direc)onality?
• All fluorescence was measured using a TECANM1000 • Fluorescence data was approximately normalized using
the K constant calculated for bidirec)onal promoters • The downstream start codon coding for sfGFP is
preferred • The downstream start codon was also recognized and
translated at a lower rate
• RFP varia)ons correspond to level of codon op)miza)on • ACG is the op)mized codon for threonine in DH10B E.coli • Constant levels of GFP demonstrate that enough charged tRNA is available to con)nue protein synthesis in the cell • Designed and implemented a gene)c circuit which tests codon op)miza)on, using threonine as an example
• All fluorescence was measured using TECANM1000 • Promoter can promote in both direc)ons • Palindromic promoters were not expected to have a bias, but
were shown to promote primarily in one direc)on • Designed and implemented a gene)c circuit to test
direc)onality of promoters
RFP GFP
Method • Circuit was generated using Gibson Assembly • Promoters were ligated between reporters • Promoter strength and direc)onality correspond to measured fluorescence
Design
Figure 1: GFP and RFP Fluorescence of different promoters
Figure 2: Fluorescence of first Start Codon (RFP) and second Start Codon (sfGFP)
Figure 3: Codon Bias in DH10B E. coli for Threonine. GFP is a constant background, while mCherry is the protein produced with the repeat codons.
ACT ACC ACA ACG
RFP GFP
Results
GFP-‐RFP Normaliza&on
• Normaliza)on of RFP and GFP fluorescence was possible though a calculated constant K • Direc)onality value was calculated using the normalized GFP/RFP ra)o
Design ORF (+1)
ORF (0)
• Circuit was generated using Gibson and PCR assembly • Design allows for uninhibited protein transla)on through both ORFs • Each start codon corresponds to a fluorescent protein in its reading frame
Method
Fluorescence of RFP and GFP Promoters
1
sfGFP RFP
Results
Design
Method
• Circuit was generated using PCR assembly • Repeated sequences were designed and inserted • mCherry is the repeat-‐sequence reporter • GFP is the control reporter
Fluorescence of RFP and GFP Start Codons
Results
Rela)ve flu
orescence
Rela)ve flu
orescence
Threonine codons
Threonine 6-‐Repeat Codons Sequence Threonine Codon ACT 5’ – GATCCACTACTACTACTACTACTA– 3’
Threonine Codon ACC 5' – GATCCACCACCACCACCACCACCA – 3’
Threonine Codon ACA 5' – GATCCACAACAACAACAACAACAA – 3’
Threonine Codon ACG 5' – GATCCACGACGACGACGACGACGA – 3’
Human Prac)ces Penn State iGEM visited a local high school and presented the background, design, data, and results from this year’s project. Students were taught the basic terminology and techniques of synthe)c biology.
A publicly accessible anima&on created by a team member was uploaded to YouTube, in addi)on to each project descrip&on presenta&on shown to the students.
Penn State iGEM members also par)cipated in a presenta&on and Q&A session with prospec&ve high school iGEM par&cipants during the Americas East Regional Jamboree
Bba_J23102
Palindrome 1
Palindrome 2
Palindrome 3
Bba_J23114
RFP GFP