Team:NYU Gallatin/Project/Cloning

From 2012.igem.org

Cloning

Project

Designer Suzanne Lee showed that bacterial cellulose could be used in novel ways, including to make clothing using a more eco-friendly process. Our team set out to demonstrate that new characteristics could be added to the cellulose produced by Acetobacter xylinum using synthetic biology methods. Altering physical characteristics such as strength, color, odor, etc. would result in exciting new materials to create with.

Cellulose is a polymer made up of long β-1,4 glucan chains. In Acetobacter, the enzyme cellulose synthase catalyzes the biosynthesis of cellulose from UDP-glucose. We hypothesized that mixed polymers containing different sugars would have unique physical properties. Cellulose synthase has been reported to utilize UDP-N-acetylglucosamine (NAG) molecules (components of chitin) as well, resulting in a polymer containing both glucose and NAG units- a cellulose-chitin hybrid. We wanted to test the properties of this new material, so we set out to engineer Acetobacter to produce this hybrid polymer.

Strategy

The yeast Candida albicans produces chitin and uses it in spore formation. To incorporate NAG into Acetobacter cellulose, we focused on three yeast genes (the pathway consisting of NAG5 (GlcNac kinase) catalyzes the conversion to GlcNac-6P, AGM1 (Phosphoacetyl-glucosamine mutase) which converts GlcNac-6-P to GlcNac-1-P, and UAP1 (UDP-GlcNac pyrophosphorylase) which adds UDP.

The most efficient method was to use Gibson assembly to piece together each gene from small, ovelapping fragments of between 300 and 500bp ("G-blocks") and clone it into pSB1C3 separately. We could not construct the entire pathway with G-blocks because it was too large, and there is a limit of six fragments for efficient Gibson assembly. The coding sequences were modified to reflect codon usage in Acetobacter, and an Acetobacter RBS was added in front of each gene. The G-blocks that we had synthesized for each gene are shown below:

  • G-blocks for AGM1
  • G-blocks for NAG5
  • G-blocks for UAP1

This worked very well, and resulted in the submission of three new parts to the BioBrick Library (Bba_K850000, Bba_K850001, Bba_K850002). We then set out to fuse these three genes into a single pSB1C3-based plasmid. PCR primers were synthesized to bracket each of the three genes and also add sequernce that would facilitate Gibson assembly of the entire pathway. Our reasoning was that we could then use Gibson assembly to piece together the three genes in the pathway together into pSB1C3. The sequences of these primers is shown below. We included two forward primers dfor the AGM1 gene- one that included the T7 promoter (known to work in Acetobacter, whose own promoters are not clearly understood yet), and one that did not.

 

Primers

  • Forward AGM1 (biobrick plasmid + promoter
  • Reverse AGM1
  • Forward NAG5
  • Reverse NAG5
  • Forward UAP1
  • Reverse UAP1 (biobrick plasmid)

The primers for the NAG5 and UAP1 genes worked well, resulting in PCR products of the expected size. However, the AGM1 primers did not result in a PCR product even when used under a variety of conditions. We then altered our cloning strategy and attempeted to use traditional BioBrick assembly. The plasmid containing AGM1 (Bba_K850000) was digested with Spe1 and Pst1, and the product gel-purified and treated with Antarctic phosphatase. The NAG5 gene was cut out of the Bba_K850001 plasmid using Xba1 and Pst1 and gel-purified. These two pieces of DNA were ligated together to produce a plasmid containing the AGM1 and Xba1 parts of the pathway in a BioBrick format. This construct was then cut with Spe1 and Pst1, and the process repeated with the UDP1 gene cut from the Bba_K850002 BioBrick plasmid with Xba1 and Pst1.

This resulted in a plasmid containing the entire pathway (AGM1, NAG5 and UDP1) but no promoter. To test the pathway in Acetobacter, we cut it out of the BioBrick vector using EcoR1 and Pst1 and cloned it into the MCS of pUC18 which has a T7 promoter.

Learn how to design primers from Julie Wolf.

Protocols

Here are some protocols we used in our cloning process.

Gibson Assembly






Chitin Synthase Mutagenesis

Amplification of CSH3 gene encoding chitin synthase from S. cerevisiae

We are also setting out to clone the chitin synthase of S. cereivisiae that was originally submitted as a part but is currently not BioBrick compatible (Part:BBa_K418007).

PCR primers were designed for amplifying CSH3 from colonies of S. cerevisiae. Primers had BioBrick prefix and suffix ends. The suffix end also had a terminal XhoI site for cloning into the pET28-b+ expression vector for His tagged.

Forward-CSH3 (BioBrick Prefix)
GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAC CGG CTT GAA TGG AG

Reverse-CSH3 (BioBrick Suffix)
GTT TCT TCC TCG AGC TGC AGC GGC CGC TAC TAG TAT TAC TAT GCA ACG AAG GAG TCA C

As this gene has 2 illegal Xba1 sites and and illegal Pst1 site, 3 sets of primers will be designed to introduce point mutations in order to remove them, to ensure BioBrick compatibility. For this strategy we will use a PCR based mutagenesis system.

Illegal XbaI sites and PstI site in CSH3 as determined using NEBcutter:

Cut pos. MS Enzyme               Recognition sequence
-------- -- -------------------- --------------------
    1117    XbaI                  T^CTAG_A
    1674    XbaI                 T^CTAG_A
    2322    PstI                    C_TGCA^G

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