1) Researchers created a cloning vector for the methanogen Methanosarcina acetivorans C2A to measure gene expression. The vector included the lacZ gene and native pmcrB promoter but was uncertain about the origin of replication. 2) Two cloning methods were attempted - Gibson assembly and PCR sewing with restriction enzymes. Gibson assembly failed to ligate correctly while PCR sewing excluded a small DNA segment but maintained the correct sequence otherwise. 3) Beta-galactosidase assays will be used to quantitatively measure protein expression of the introduced lacZ gene. Preliminary assays showed the method works for a shuttle plasmid and that the native methanogen has no background lacZ activity. The recombinant plasmid created

1. Introducing the lacZ Gene into Methanosarcina acetivorans C2A to Measure pmcrB Promoter Activity
Eduardo Perez, Ann Lesnefsky, Alfred Spormann
Stanford University
Introduction
There is an need to create organic compounds in a carbon neutral way to
prevent CO2 from accumulating in the atmosphere and in turn prevent
global warming. One method of producing organic chemicals from CO2
is using anaerobic microorganisms. Methanosarcina acetivorans C2A is
an anaerobic microorganism that reduces CO into methane and other
organic compounds, but at a slow rate. This work focuses on tools to
have control over gene expression of M. acetivorans C2A which can be
used to manipulate anaerobic metabolism and in turn produce more
organic compounds.
Background
Previous attempts to understand gene expression for Methanosarcina
species have studied promoter libraries in an in vitro setting. This work
looks at gene expression in M. acetivorans C2A in vivo. A promoter
library will be tested in vivo by measuring the protein activity of the
expressed lacZ gene using the beta-galactosidase assays. LacZ is present
in E. coli MG1655 and in plasmid pUC19, but it is not native to M.
acetivorans C2A.
Method Overview
In order to measure the activity of the lacZ gene in
the methanogen, the gene needs to be inserted into
the methanogen through a cloning vector. To
accomplish this, the native pmcrB promoter, and the
methanogen origin of replication need to be included
in the cloning vector.
The cloning performed followed the typical
procedure. The vector with a multiple cloning site
(1) and Insert (2) were digested, then annealed to
create a recombinant plasmid.
The plasmid was then transformed into E. coli.
The transformants were selected using appropriate
antibiotics.
Results
1. Alper, Hal, Curt Fischer, Elke Nevoigt, and Gregory Stephanopoulos. "Tuning Genetic Control through
Promoter Engineering." Proceedings of the National Academy of Sciences of the United States of America
102.36 (2005): 12678-2683. Web. 17 Sept. 2015.
2. Guss, Adam M. et al. “New Methods for Tightly Regulated Gene Expression and Highly Efficient
Chromosomal Integration of Cloned Genes for Methanosarcina species.” Archaea 2.3 (2008): 193–203.
Print.
3. Soppa, J. (1999), Transcription initiation in Archaea: facts, factors and future aspects. Molecular
Microbiology, 31: 1295–1305. doi: 10.1046/j.1365-2958.1999.01273.x
Conclusion
The cloning vector created for M. acetivorans C2A includes the lacZ
gene along with the native pmcrB promoter, but uncertain about origin of
replication. It will be transform it into the methanogen to see whether the
mutation excluded it or not. After the transformation, an assay needs to
be performed to see the protein expression of the lacZ gene in the
methanogen. Future work will involve mutating the promoter sequence
and measuring protein expression.
References
In Gel Ligation Cloning
The second method used to create the cloning vector involved PCR
sewing with restriction enzymes. The plasmids used are also pWM321
and pCDF.
Gibson Assembly Cloning
The first method to create the cloning vector involved Gibson assembly.
This process took the lacZ gene and linked it onto the pWM321 vector
backbone. The process was done because it does not rely on restriction
Figure 3. Gibson assembly mechanism. Igem.org
The Gibson assembly did not ligate correctly. Sequencing results had
excluded DNA segments, possibly removing origin of replication.
Highlighted is where the mutations begin.
Methods
After different attempts with
various restriction enzymes,
the correct digest was
achieved with KpnI and SalI.
The highlighted bands in the
gel image were then cut out
by hand in 0.8% low melting
agar gel and annealed.
The resulting DNA was then
transformed into E. coli cells
and grown to replicate the
plasmid.
1
2
enzymes, eliminating
possible errors in
digestion.
PCR products are
created using primers
and annealed using
NEB Gibson Assembly
Mix.
Beta-Galactosidase Assay
Beta-galactosidase assays are performed to quantifiably measure protein
expression of the induced lacZ gene. This method has worked on
methanogens in previous experiments because it deals with lysed cells in
an aerobic setting, eliminating the need for an anaerobic environment.
The experiment used E. coli DH5α
λpir with and without pUC19, E. coli
MG1655, and a blank. The plasmid
pUC19 is a shuttle plasmid with lacZ
gene. If the assay worked for this
shuttle plasmid, there is reason to
believe it will work for transformed
M. acetivorans C2A.
The experiment was also performed
with M. acetivorans C2A to check
background activity.
In gel ligation results were more promising. Although it excluded a 30
base pair sequence, it maintained all other correct sequencing. Due to
uncertainty on where the origin of replication is located, this
recombinant plasmid will be transformed into M. acetivorans C2A.
Results from the first assay demonstrated that it could successfully be
replicated in the lab.
Results from the second assay demonstrated native M. acetivorans C2A
did not have background activity.
The lacZ gene is induced by
the presence of IPTG.
Without IPTG, the lacZ
promoter does not express
the lacZ gene.
Figure 1. openwetware.com
Figure 2. molecularhub.com