2. Ligation reactions were carried out using 50 ng of vector DNA
and an insert to vector ratio of 5:1. The temperature at which
the reactions were carried out was generally between 20–25
C.
3. A conventional chemical transformation procedure was performed to transfer these constructs during this process, but
larger single plasmids or multiple plasmids might require an
electrotransformation process. The plasmids presented in this
method either contain one specific antigen (the pCJ or pUAB)
or the combined antigens (pLF).
4. An array of metal-chelation affinity chromatography options
exist for purifying 6Â histidine-tagged recombinant protein
products. In this case, we used a HisTrap column system that
relied on NTA-Nickel chelation.
5. It is common to concentrate or evaporate the solution to the
densest level possible. In doing so, we observed no component
breakdown at 35
C.
6. At OD 600nm 0.4–0.5, E. coli is at a period of exponential
growth when the proliferation rate is at the highest level, likely
resulting in cellular health supporting optimal gene expression.
7. IPTG is used to induce the expression of genes in the plasmid
or genome in E. coli. Normally, the induction temperature
should be less than 30
C and induction time depends on the
extent of gene expression desired (i.e., how much protein is
required for a given dose).
8. The NaOAc solution provides an environment that facilitates
polymer coating onto the E. coli surface.
9. Shaking at 22
C helps the polymer coating process. PBS is
applied during dilution to maintain cellular health and pH.
10. Sufficient monitoring and timely humane euthanasia when
mice are observed to be experiencing distress is critical for
ethics in conducting studies with vertebrate animals.
Acknowledgments
The authors recognize support from NIH awards AI088485 and
AI117309 and a grant from the Technology Accelerator Fund from
the State University of New York Research Foundation (all to B.A.
P.).
References
1. Harris CC (1991) Chemical and physical carcinogenesis: advances and perspectives for the
1990s. Cancer Res 51(18, Suppl):5023s–5044s
2. Jones CH, Chen M, Ravikrishnan A,
Reddinger R, Zhang G, Hakansson AP, Pfeifer
BA (2015) Mannosylated poly(beta-amino
esters) for targeted antigen presenting cell
474
Ruiquan Qi et al.
and an insert to vector ratio of 5:1. The temperature at which
the reactions were carried out was generally between 20–25
C.
3. A conventional chemical transformation procedure was performed to transfer these constructs during this process, but
larger single plasmids or multiple plasmids might require an
electrotransformation process. The plasmids presented in this
method either contain one specific antigen (the pCJ or pUAB)
or the combined antigens (pLF).
4. An array of metal-chelation affinity chromatography options
exist for purifying 6Â histidine-tagged recombinant protein
products. In this case, we used a HisTrap column system that
relied on NTA-Nickel chelation.
5. It is common to concentrate or evaporate the solution to the
densest level possible. In doing so, we observed no component
breakdown at 35
C.
6. At OD 600nm 0.4–0.5, E. coli is at a period of exponential
growth when the proliferation rate is at the highest level, likely
resulting in cellular health supporting optimal gene expression.
7. IPTG is used to induce the expression of genes in the plasmid
or genome in E. coli. Normally, the induction temperature
should be less than 30
C and induction time depends on the
extent of gene expression desired (i.e., how much protein is
required for a given dose).
8. The NaOAc solution provides an environment that facilitates
polymer coating onto the E. coli surface.
9. Shaking at 22
C helps the polymer coating process. PBS is
applied during dilution to maintain cellular health and pH.
10. Sufficient monitoring and timely humane euthanasia when
mice are observed to be experiencing distress is critical for
ethics in conducting studies with vertebrate animals.
Acknowledgments
The authors recognize support from NIH awards AI088485 and
AI117309 and a grant from the Technology Accelerator Fund from
the State University of New York Research Foundation (all to B.A.
P.).
References
1. Harris CC (1991) Chemical and physical carcinogenesis: advances and perspectives for the
1990s. Cancer Res 51(18, Suppl):5023s–5044s
2. Jones CH, Chen M, Ravikrishnan A,
Reddinger R, Zhang G, Hakansson AP, Pfeifer
BA (2015) Mannosylated poly(beta-amino
esters) for targeted antigen presenting cell
474
Ruiquan Qi et al.