1. BioSketch The bacterial sketch pad.
Yves Wang, Jennifer Gao, Yin Li
Chris Doucette, Thomas Noriega, Hing Eng
Group Meeting

2. Talk outline What has been done with the Collins constructs
What has been done with the BioBricks
What’s in store for this week

3. Collins (Yin and Jenny)

4. What has been done with Biobricks
Pairwise assembly:
QPI digestions successful with modified protocol:
Pl + QPILac
QPIl + YFP
QPI434 + YFP
Completed first two rounds and started third round of ligations to build QPIs from scratch
Performing ligations in duplicate to decrease chances of setbacks
Sequenced successful ts (mut265) mutations in LacI
Have sent LacI ts (mut241) and both QPILacI(ts) mutants (m241 and m265) for sequencing
Outsourced 2 different ts, ind- versions of lcI. (“mJP3” based on Japanese patent Genbank #E07700, and “m8572” based on cI857 sequence found in HENDRIX Lambda II book).

5. Ligations Successful Ligations: Digestions ready for ligation:
mCherry + Terminator
RBS + 434
RBS + lcI
RBS + mCherry-Terminator
RBS + Venus-Terminator
Digestions ready for ligation:
PlambdaCI
P434
PlacI
PlacI(hybrid)
434
lcI
RBS-434
RBS-lcI
RBS-mCherry-Terminator
RBS-Venus-Terminator
QPIlacI
QPIl
QPI434

6. Summary: What we have done
CollinsMod: (?)
BioBricks:
Pairwise assembly
mCherry + T
RBS + 434
RBS + lcI
RBS + mCherry-Terminator
RBS + Venus-Terminator
QPI digest problem solved; ligations still in testing
Mutations and Sequencing
We have a sequenced Bb-LacI m265
We are sequencing LacI m241 and QPI-lacI m241 and m265
Introducing mJP3 and m8572 into Lambda cI and QPI-Lambda cI

7. This week with Collins

8. This week with Biobricks
More Pairwise Assembly
First Round
PlacI(hyb) + RBS-lCI
Pl + RBS-434
RBS + LacIts(265)
All promoters + mCherry
All promoters + Venus
Second Round
Terminator + P434-mCherry
Terminator + P434-Venus
Terminator + Pl -mCherry
Terminator + Pl -Venus
Sequence cI857 mutations
Test microscopy with Promoter-Reporter constructs
Are mCherry-expressing colonies red enough?
Make Tsien lab bacterial mCherry into BioBrick
QPIl + mCherry
QPI434 + mCherry
Pl + QPIlacI
Pl-QPIlacI + QPIl-mCherry
Pl-QPIlacI + mCherry
PlacI + QPIl-mCherry
Pl + QPI434-mCherry