Team:BIT-China/Design
Design
What is the problem?
Plasmid segragational instability has been the limit
step for large-scale protein production and bioremediation.
Use of antibiotics for plasmid retention is not practical
in this situation.
High copy number (hcn) plasmids are lost from population at a high rate due to the
large metabolic burden and lack of nature factors for plasmid maintenance.
Our P-SLACKiller
IDEA
Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller). Use the inhibitor protein as a signal indicating plasmid numbers,
when the plasmid concentration reduces to a threshold, the switch of killer gene will turn on.
PART DESIGN
The basic circuit is shown in Fig.2, the genetic locus of functional gene can be substituted on the basis of production demand in the future. Inhibitor and in-promoter are key to control the threshold.
When the signal of plasmid losing is detected, the executor killer will play a role.
Killer device
As an executor for killing the cell labeled as slackers, killer device
should include the detecting part like in-promoter interacting
with inhibitor protein as well as the killing part like toxin gene
mazF and hokD.
Ways of self-killing:
1) toxic protein
2) sgRNA targeting genome of strains with no NHEJ repair system
3) sgRNA targeting essential genes
PART DESIGN
At the first stage, we need to verify the function of two toxin proteins
and make sure the bacteria can be killed after arabinose induction.
To control the expression of killer gene, instead of J23119,
arabinose inducible regulatory promoter/repressor unit [part number] is employed in our circuit.
After measuring the growth curve, we observed the obvious
difference between the testing group and control group. [see results page for killer]
To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with
the killer circuit and replace the RFP for killer gene. [see design for threshold]
For the toxic protein,
MazF and HokD were chosen as candidates. [see more about the killing mechanisms]
Threshold device
Inhibitor and in-promoter are used to measure and adjust
the threshold. Before apply the threshold value into our
model, we need to use RFP as a reporter
gene since the expression of toxin protein is hard to quantify. (know more about our model)
Goal
(1) Build three parallel circuits employing different inhibitor proteins
(2) Find out the initial thresholds by measuring RFP intensity
(3) Provide experimental data for modeling part
Part Design
The gene circuits are shown in Fig.5.
(1) The concentration of inducer arabinose is regarded as an input signal,
thus Part:BBa_K808000 Arac-pBAD is used to control the expression of inhibitor proteins
(2) Three combinations of inhibitor and in-promoter are selected to prove
the concept of plasmid quorum sensing [see inhibitor mechanisms]
(3) RFP is used as an indicator
We assume that:
The high concentration of inducers can simulate high concentration
of plasmids due to the same effect of producing more inhibitor proteins.
By adding different concentration of arabinose, we can measure the fluorescence intensity
of RFP and find out the initial thresholds under control of certain inhibitor proteins.
Upgraded P-SLACKiller
Why do we need to optimize our project?
It’s possible to produce generation of plasmid-free cells.
Thus, the killer device should be integrated into the genome. [see reombination]
To meet various needs with different plasmid numbers,
we need to adjust the initial threshold
through replacement of RBS as well as in-promoter mutation. [see mutation]
Recombination
Recombineering (recombination-mediated genetic engineering) is
an efficient molecular engineering technique used for gene
replacement, deletion and insertion.
Ways: a linear plus circular reaction
A：Employment of traditional λ-Red recombination
PART DESIGN
The basic circuit of upgraded P-SLACKiller is shown in Fig.6.
Compared with the original one, two homologous arms (50bp) are added.
With the help of lambda Red recombination system, the killer part can be integrated into genome,
preventing the whole system from being invalid. The final gene circuit is shown in Fig.7.
B:：CRISPR-Cas9 coupling λ-Red recombineering
PART DESIGN
Difference between the two methods above: Traditional way use the resistance
gene as a selection marker, while the CRISPR coupled way
introduce a double-strand break (DSB) into the strain with no recombination occurred.
Application:
In our project, we tried both the two ways to test the integration
efficiency. However, the CRISPR coupled
way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction.
We tried the traditional way with the pKD46 plasmid
provided by Chun Li’s lab and got the positive results. [results for recombination]
Two main factors will influence the recombination efficiency: the target site on genome
and the length of homologous arms between donor fragment. [recombination protocol]
Promoter mutation
For inductive promoters, two kinds of region are significant
to its transcription strength. One is common -10 and -35 region
which can firmly bind to RNA polymerase, the other is
operon region binding with regulatory factors like
inhibitor proteins. [see mutation mechanisms]
Aim:
(1) To adjust threshold through changing in-promoter’s strength
(2) Tied to specific promoter like pTet, build a mutants library and improve the existing part by measuring the strength through RFP
Part Design
After analyzing the promoter region of pTet, we designed
random primers to mutate its -35 region.
RFP intensity is measured to indicate the strength of
mutated promoters. [see mutation results]
Design
