Team:Warsaw/results

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Project results

In this place we want to share results we have obtained during our Project. Although we haven't managed to finish everything we have planned to, we can be really happy of some partial results, which are described below.


Contents


lacI/cI bistable switch

Control of cell invasion and further operation of our system - the BacInVader, is done by two bistable switches. The first one controls expression of invasion genes.

Design

The structure of this switch is shown below:

LacI
R0010

B0032
cIts
K177050

B0030
GFP
E0040

B0010

B0012
cI lam
R0051

B0032
lacI
C0012

B0034
mRFP1
E1010

B0010

B0012

The most important components of the switch are repressor proteins cI (denoted here as cIts, see explanation below) and lacI. The whole device is composed of two main parts:

LacI under control of 'cI lam' promoter

cI lam
R0051

B0032
lacI
C0012

and

cI under control of Plac promoter

LacI
R0010

B0032
cIts
K177050

How it works?

Because cI is repressor of 'cI lam' promoter and lacI is repressor of Plac promoter (denoted here as 'LacI') there are only two mutually exclusive states of the switch:

  • Plac promoter is active which leads to cI expression. CI binds to 'cI lam' promoter and represses it so no genes under this promoter are expressed.
  • 'cI lam' promoter is active which leads to LacI expression. LacI binds to Plac promoter and represses it.

According to this, state of the switch should be stable in time until some external stimulus is applied.

How to switch its state?

To set the state of the switch 0,1mM IPTG and high temperature (42oC) are used.

  • After addition of 0,1mM IPTG LacI gets inactivated and transcription from Plac starts. cI is expressed which leads to repression of 'cI lam' promoter.
  • Heating the bacteria in 42oC inactivates CI protein and starts expression from 'cI lam' promoter.

Selection of strains compatible with switch

Because LacI interferes with operation of our device we needed E. coli strain that is LacI deficient. To choose such strain we have constructed lacI detector device BBa_K177024:

LacI
R0010

B0030
GFP
E0040

B0010

B0012

It's simply GFP with strong RBS under lacI repressed promoter. Without IPTG expression of GFP is LacI dependent. In LacI deficient strain GFP is expressed. When cells express LacI it binds operator site in promoter sequence wihch leads to no GFP expression.

We have tested following strains: Top10, Top10 F' (control strain because it contains additional copy of LacI), DH5alpha, GM2163, BL21, BBa_V1011 and BBa_V1012. All strains were grown in liqiud medium with and without 0,1 mM IPTG. Strains which expressed GFP regardless of addition of IPTG were found to be lacI deficient.

Results: (expression of GFP without IPTG/with IPTG/conclusion)

  • Top10 F': -/+/lacI proficient, control ok
  • Top10: +/+/lacI deficient
  • DH5alpha: -/+/lacI proficient
  • BL21: +/+/lacI deficient
  • GM2163: +/+/lacI deficient
  • BBa_V1011: -/+/lacI proficient
  • BBa_V1012 -/+/lacI proficient

Top10 strain was chosen for further expreiments.

Experimental results

To test if our assumptions are correct we have assembled switch testing device BBa_K177038. In this device we have connected GFP with Plac dependent part of the switch and mRFP with 'cI lam' dependent part. After transformation of the complete construct at 30oC random distribution of green and red colonies was observed:

Switch-unset.jpg

It was expected because we haven't used temperature nor IPTG to set the initial state of the switch.

We have chosen red (R) and green (G) colonies which served us as starting point for further experiments and cultured them on plates:

Switch-start.jpg

Then we have cultured green colonies in liquid culture and on plates in 42oC and they started to express RFP:

Switch-red-plate.jpg Switch-red-lc.jpg


Red colonies were grown in liquid culture and on plates in 30oC with 0,1mM IPTG and they turned green (GFP+ phenotype):

Switch-green-plate.jpg Switch-green-lc.jpg


It indicates that our lacI/cI bistable switch works as expected.

To test stability of the states both red and green colonies were grown in liquid culture in 30oC. The 'red' ('cI lam' promoter active) state remained stable for about 9 hours. After that time cI repressor took over and bacteria started to express GFP. The 'green' (Plac promoter active) state was stable after overnight culture. Spontaneous switching from red to green wasn't observed on plates, where incubation period of 36 hours didn't lead to change of state.

We conclude that level of LacI production may be to low to ensure complete repression of Plac when cells are growing rapidly in liquid medium. This results in 'leaking' of Plac promoter and accumulation of CI protein which eventually leads to state switching. Improved versions of this device should have stronger RBS before lacI and/or use Plac promoter with more lac operator sequences to ensure that leaking of Plac will be minimized.

Bistable switch parameters

Current version of our cI/lacI bistable switch has following parameters:

  • Temperature of stable state: 30oC
  • Strain requirements: strain should be lacI deficient
  • Plac->'cI lam' switching: in 42oC
  • 'cI lam' -> Plac switching: in 0,1 mM IPTG
  • State stability in plate culture: >36h for both states
  • State stability in liquid culture: ~9h for 'cI lam' driven (red) state; >18h for Plac driven (green) state

Termosensitive version of lambda cI protein

The very first version of switch testing device BBa_K177038 was using cI lambda repressor form the Registry BBa_C0051. It behaved oddly - nearly all colonies on plates were red and those very few that were green turned spontaneously red in liquid culture. Addition of IPTG resulted in pink (both GFP+ and RFP+) colonies.

It lead us to conclusion that our copy of BBa_C0051 was somewhat defective and wasn't able to repress 'cI lam' promoter efficiently enough.

We have sequenced this brick (results here: File:BBa C0051-Warsaw.txt, also submitted to the Registry) and it turned out that it has point mutation C->T at position 450 of cI CDS which leads to formation of premature stop codon at amino acid position 360. Additionally it has two other point mutations S504->N and S786->C.

This version of cI even after fixing of premature stop has only moderate thermosensitivity so we have decided to introduce another point mutation - cI857ts together with fixing premature stop. This lead to creation of our cIts Biobrick BBa_K177050.

It works as expected repressing lambda PRM promoter and derivatives (in case of our switch BBa_R0051). Additional thermosensitive mutation - cIts857 causes deactivation of our cIts repressor in 42oC which was ideal for our purposes. It allowed us to control behavior of bistable switch making BBa_K177038 a functional part.


What is remaining to be done

To get fully functional BacInvader system we need to obtatain some more bricks. At first - invasin (BBa_K177010) and/or inernalinA (BBa_K177032) are needed for effective bacteria invasiveness.

In the next step, endosome detection operon, we have some technical problems with cloning of PhoP/PhoQ genes (BBa_K177007) and promoter of MgtC gene (BBa_K177021) from Salmonella enterolitica ser. typhimurium. We have got proper PCR products but there are some difficulties with cloning. When obtained, this parts will be interesting sensor of low pH conditions.

Another thing is preparation of bioBricked secretion system and mitochondrial targeting signal from the Cox gene (BBa_K177028).

Some of our further plans are also described here.