Construction and Testing

From 2007.igem.org

Contents

Construction Tree

The diagram below outlines our parallel construction plan, from individual registry parts to the final half-adder. Because many people were working on the project at once, it is designed to keep everyone organised.

Construction Chart

Diagram details:

  • Expected fragment sizes are shown at every node so gel results can be analyzed quickly.
  • Vector resistances for each part/construct are shown, with blue representing ampicillin resistance and red representing kanamycin resistance. Purple represents a resistance to both.
  • Parts and developing constructs all have abbreviated names for ease of labeling. To make the subtle distinction between a coding sequence and a coding sequence with a ribosome binding site, we use a ' on the computer and an underline when writing. A double ' or double underline indicates a coding sequence with a ribosome binding site and a transcriptional terminator.
  • Arrows show the direction of DNA cloning and specify which restriction enzymes to use for cutting. Hollow arrows are present when two parts are ligated from vectors of the same resistance, requiring additional methods such as the use of alkaline phosphatase, gel extraction, or a three-way ligation into a vector with different resistance.
  • The numbers 1-2-3-3.5-4 indicate which major protocols need to be carried out at each step in the assembly cycle:

1) Transform selected DNA.

2) Streak colonies from transformation spread plates. Patch on to plates of the opposite resistance. Innoculate culture tubes.

3) Use the patches to discard colonies which are incorrect. Miniprep from the culture tubes of potentially correct colonies. Digest DNA with NotI and run on a gel. Discard colonies which have incorrect sizes. Innoculate tubes from correct colonies.

3.5) Perform a full digest using appropriate enzymes on correct parts. Ligate parts together.

4) Create a glycerol stock from the innoculation.

Testing Constructs

We use the fluorescent proteins GFP and RFP as reporter genes to test the functionality of some of the components and constructs of our half-adder design. Where possible, we make use of the lac inducible promoter to control expression of the enhancing or repressing elements.

Comparing basal and induced expression levels for the quorum sensing promoters

CONSTRUCT AIMS
UW testing constructs1AB.png The quorum-sensing promoters: How active is 'unactive'?
  • Preliminary test for the basal levels of activity of the positively-controlled quorum sensing promoters, Plux and Plas
UW testing constructs1CD.png The quorum-sensing constructs: How off is 'off' and how on is 'on'?
  • Test for levels of expression in the unenhanced versus enhanced state.
  • lasR/luxR must be expressed in same strain as Plas/Plux (respectively) since these genes encode transcription activators regulated by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes.
  • The two strains are grown in a single culture tube for this test.


Testing functionality of the various promoters

CONSTRUCT AIMS
UW testing constructs2A1A2.png The Omp promoter
  • Positively-controlled promoter activated by endogenous OmpR protein (sensitive to salt concentrations via EnvZ protein)
  • Test for activity using high salt levels in EnvZ+ strain
  • Test for activity using the photo-active red system in EnvZ- strain
UW testing constructs2B.png The Tet promoter and operator
  • Negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
  • Test for levels of expression during repressed versus derepressed state
UW testing constructs2CD.png The Plas and Plux quorum-sensing promoters
  • Test for levels of expression in the unenhanced versus enhanced state
  • lasR/luxR must be expressed in same strain as Plas/Plux since these genes encode transcription activators regulated by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes
  • The two strains are grown in a single culture tube for this test
UW testing constructs2E.png The PlasOcI promoter-operator fusion
  • Both negatively- and positively-controlled promoter in the final half adder design, repressed by cI and enhanced by LasR
  • Test for activity in the repressed versus enhanced state
  • Test in addition to above construct since cI operator fusion may affect las promoter activity


Parts Submitted to the Registry

The following is a table listing all of the parts submitted to the registry.

Name Part Number Constructs Comments
rfp' BBa_S03873 UW 1.PNG confirmed (functional after promoter)
ho1' BBa_S03877 UW 3.png
lasI' ' BBa_S03878 UW 4.PNG LasI protein generator
lasR' ' BBa_S03882 UW 14.PNG LasR protein generator
pcyA' BBa_S03874 UW 12.PNG
pcyA' ' BBa_S03875 UW 2.PNG PcyA protein generator
tetR' BBa_S03879 UW 13.PNG
tetR' ' BBa_S03880 UW 5.PNG TetR protein generator
Plas rfp' BBa_S03885 UW 8.PNG
Plac lasr' ' BBa_S03883 UW 7.PNG
Ptet rfp' BBa_S03887 UW 9.PNG functionality confirmed
Pomp rfp' BBa_S03888 UW 10.PNG functionality confirmed
Plux rfp' BBa_S03889 UW 11.PNG functionality confirmed
PlasOcI rfp' BBa_S03881 UW 6.PNG functionality confirmed

Notes:

  • ' denotes that the construct contains the ribosome binding site (RBS) and the coding region for that gene.
  • ' ' denotes that the construct contains the ribosome binding site, coding region for that gene, and a transcriptional terminator. 


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