Cyanobacteria:

1
Cyanobacteria

• Week 8

2
Outline

• Project Goal
• Extracting and biobricking KaiA and KaiB
• Synthesis update
• Western Blotting update
• Site-Specific Mutagenesis update
• Promoter Choice
• Future Plans

3
Project Goal
Reconstitute the cyanobacteria KaiABC oscillator
in E. coli.

• Create KaiA, KaiB, and KaiC biobricks.
• Transform E. coli with Kai Biobricks to
  reconstitute KaiC phosphorylation cycle (no
  reporter attached).
• Distant Transform E. coli with Kai Biobricks to
  reconstitute KaiC phosphorylation cycle with
  Biobrickd reporter.

4
Clarification

• We now have 3 different methods for getting our
  KaiA, B, and C in a biobrick
• Extraction of Kai A, B, and C separately w/ BB
  ends
• Attempted this week
• Synthesis of Kai A, B, and C separately w/ BB
  ends
• A cassette of Kai A, B, and C w/ BB ends
• Attempted past month
• We have KaiA, and KaiB we will be getting KaiC
  at the end of the week.

5
Kai BioBricks

• Performed a PCR to extract kaiA, kaiB, and kaiC
  sequences individually (with BioBrick ends)
• We successfully extracted kaiA and kaiB, but not
  kaiC
• Performed a digestion of KaiA, KaiB, and a
  digestion of RFP-containing plasmid (Bba_J04550)
• Performed a ligation/transformation of the
  RFP-containing backbone with kaiA and kaiB
• Currently growing up the transformants

6
Synthesis Update
kaiB
kaiA
367 bp In standard vector
913 bp In standard vector
7
Western Blotting Update

• Susan Golden expressed willingness to send us
  antibodies for KaiC
• Let me know how many westerns you think you want
  to run, and we'll send you some antiserum. I am
  pretty sure the anti-KaiC was made in chickens--
  an important point for ordering secondary
  antibodies for visualization.

8
Site-Specific Mutagenesis update

• We received sequencing results for our
  templates cloned into BLUNT TOPO which we had
  been using for the past month
• Of 6 clones, 4 were background for the blunt
  TOPO kit
• Other two had bad transformants
• Decided to pursue PCR off of the template
  directly instead of after cloning

9
Site Specific Mutagenesis
1
2
3
4
5
6
7
8
9
10
11

• Lanes 1, 6, 11 1kb
• Lane 2 and 4 expected 210bp segment
• Lane 3 and 5 expected 80bp segment
• Lane 8 and 10 expected 3kb segment

10
Site-Specific Mutagenesis Future Plans

• We will not pursue the mutagenesis of the Kai ABC
  cassette further unless one feels the need to
  create a cyanobacteria BioBrick.

11
Promoter Choice

• LacIQ RFP, high copy

• We are experimenting with the Lac promoter
• Lac RFP, high copy
• LacIQ RFP, high copy
• Lac RFP, low copy
• LacIQ RFP, low copy

12
Future Goals

• Kai BioBricks
• Ligate KaiA/B into BB vector for amplification
• Weve decided to wait for synthesis of KaiC
• Promoter search
• Test the tightness of the Lac promoter on a
  low-copy plasmid with/without LacIq
• Test a second promoter
• Experimental constructs
• Ligate promoters, Kai BBs
• Decide which operons will go on which plasmids
• Tradeoff between doing multiple ligations and
  multiple transformations
• Western blots
• Reply to Prof. Golden regarding antibodies and
  prepare experiments