\Section{Plasmid Map - \normalfont{p}IND4}
\label{sec:Plasmid Map - pIND4}

\begin{figure}[htbp]
	\centering
		\includegraphics[scale=0.87]{appendix/figsa/plasmid_map.png}
	\caption{pIND4 plasmid map showing important features, taken from \cite{ind2009}. Expanded section shows the cloning site - inserts must start with the ATG codon which forms part of the \textit{Nco}I restriction site. If the stop codon of the inserted gene is omitted, a C-terminal histidine tag will be added. All the pIND4 derivatives made in this work included the stop codon so no tag was added.}
	\label{fig:plasmid_map}
\end{figure}
\clearpage
\Section{Primers}
\label{sec:Primers}

In the tables below, restriction cut sites are \underline{underlined} and deliberate mutations are marked in \textbf{bold}. 

\vspace{0.5cm}
\begin{table}[htbp]
		\begin{tabular}{l l l}
		\hline
\textbf{Name}	&	\textbf{Sequence}														&	\textbf{Restriction Site}	\\
Y6-A					&	AATAC\underline{CCATGG}TGAGCAAGGGCGAGGAG		&	\textit{Nco}I		\\
Y6-B					&	CATCTCGAT\textbf{GGC}GAGCAGGAT							&	-								\\
Y6-C					&	ATCCTGCTC\textbf{GCC}ATCGAGATG							&	-								\\
Y6-D					&	AATAC\underline{GGATCC}TCAGGCGGCCATCAGCGTC	&	\textit{Bam}HI	\\
		\hline
		\end{tabular}
	\caption{Primers for making \textit{yfp-cheY$_6$}(D56A) using pIND-YFPY6 as a template. The \textit{cheY$_6$}(D56A) gene was later amplified from this plasmid to give pIND-Y6D56A using primers Y6-F and Y6-R (Table ~\ref{tab:pIND4_primers}).}
	\label{tab:D56A_overlap_primers}
\end{table}
\vspace{2cm}
\begin{table}[htbp]
		\begin{tabular}{l l l}
		\hline
\textbf{Name}	&	\textbf{Sequence}														&	\textbf{Restriction Site}	\\
Ec-Y-A					&	TACA\underline{CCATGG}CGGATAAAGAACTT			&	\textit{Nco}I		\\
Ec-Y-B					&	CTTCACCAC\textbf{CCA}GCCACTGGC						&	-								\\
Ec-Y-C					&	GCCAGTGGC\textbf{TGG}GTGGTGAAG						&	-								\\
Ec-Y-D					&	ATCG\underline{GGATCC}TCAAAATCCAAGACTATC	&	\textit{Bam}HI	\\
		\hline
		\end{tabular}
	\caption{Primers for introducing the Y106W mutation in \ec{} \textit{cheY}. The AB PCR was carried out on a pBAD-ecYD13K template. The CD PCR was amplified from \ec{} RP437 genomic DNA. The overlap PCR (AD) yields a 1053 bp product containing \textit{cheY} (including the two point mutants) and the neighbouring \textit{cheZ} gene. Following digestion, this product was ligated into cut pIND4. After sequencing the correct clone was used as a template to amplify just \textit{cheY}(D13K, Y106W) (commonly known as CheY**) with primers Ec-Y-F and Ec-Y-R (Table ~\ref{tab:pIND4_primers}).}
	\label{tab:Y106W_overlap_primers}
\end{table}

\begin{sidewaystable}[htbp]
		\begin{tabular}{l l l l}
		\hline
\textbf{Name}		&	\textbf{Target}							&	\textbf{Sequence}																			&	\textbf{Restriction Site}	\\
Ec-Y-F					&	\ec{} \textit{cheY}					&	TACA\underline{CCATGG}CGGATAAAGAACTT									&	\textit{Nco}I			\\
Ec-Y-R					&	\ec{} \textit{cheY}					&	ATCG\underline{GGATCC}TCACATGCCCAGTTTCTC							&	\textit{Bam}HI		\\
Ec-YZ-F					&	\ec{} \textit{cheY cheZ}		&	Same as Ec-Y-F																				&	\textit{Nco}I			\\
Ec-YZ-R					&	\ec{} \textit{cheY cheZ}		&	ATCG\underline{GGATCC}TCAAAATCCAAGACTATC							&	\textit{Bam}HI		\\
Ec-Z-F					&	\ec{} \textit{cheZ}					& AGCTAG\underline{GGTCTCCC}ATGATGCAACCATCAATC					&	\textit{Bsa}I			\\
Ec-Z-R					&	\ec{} \textit{cheZ}					&	Same as Ec-YZ-R																				&	\textit{Bam}HI		\\
Y1-F						&	\textit{cheY$_1$}						&	AGCTAGGGTCTC\underline{CCATG}CCGCTGACCGTTCTTGCC				&	\textit{Bsa}I			\\			
Y1-R						&	\textit{cheY$_1$}						&	AATAC\underline{GGATCC}TCACGCGACCGCCACGCG							&	\textit{Bam}HI		\\
Y2-F						&	\textit{cheY$_2$}						&	AGCTAGACCTGC\underline{ATATC}ATGGCACTCAGGGACAGCATT		&	\textit{Bfu}AI		\\
Y2-R						&	\textit{cheY$_2$}						&	AATAC\underline{AAGCTT}TCATAGAGCGCCTACGAC							&	\textit{Hind}III	\\
Y3-F						&	\textit{cheY$_3$}						&	AGCTAGACCTGC\underline{ATATC}ATGAGCAGGACGGTTCTCGCC		&	\textit{Bfu}AI		\\
Y3-R						&	\textit{cheY$_3$}						&	AATAC\underline{GGATCC}TCATCCGAGCACCTTCTTG						&	\textit{Bam}HI		\\
Y4-F						&	\textit{cheY$_4$}						&	AGCTAGACCTGC\underline{ATATC}ATGACGAAAACCGTCCTCGCA		&	\textit{Bfu}AI		\\
Y4-R						&	\textit{cheY$_4$}						&	AATAC\underline{GGATCC}TCAGCCAAGAAGCTTCTTC						&	\textit{Bam}HI		\\
Y5-F						&	\textit{cheY$_5$}						&	AGCTAGACCTGC\underline{ATATC}ATGAGCAAGACGATCCTCGCG		&	\textit{Bfu}AI		\\
Y5-R						&	\textit{cheY$_5$}						&	AGCTAGACCTGC\underline{ATATC}ATGAGCAAGACGATCCTCGCG		&	\textit{Bam}HI		\\
Y6-F						&	\textit{cheY$_6$}						&	AGCTAGACCTGC\underline{ATATC}ATGCCCTACAATGTCATGATC 		&	\textit{Bfu}AI		\\
Y6-R						&	\textit{cheY$_6$}						&	AATAC\underline{GGATCC}TCAGGCGGCCATCAGCGTC						&	\textit{Bam}HI		\\	
YFP-Y6-F				&	\textit{yfp-cheY$_6$}				&	AATAC\underline{CCATGG}TGAGCAAGGGCGAGGAG							&	\textit{Nco}I			\\
YFP-Y6-R				&	\textit{yfp-cheY$_6$}				&	AATAC\underline{GGATCC}TCAGGCGGCCATCAGCGTC						&	\textit{Bam}HI		\\
		\hline
		\end{tabular}
	\caption{Primers for cloning genes into pIND4. All genes were cloned into \textit{Nco}I/\textit{Bam}HI cut pIND4 (except for \textit{cheY$_2$} which was cloned into \textit{Nco}I/\textit{Hind}III) cut pIND4. Note \textit{Bsa}I	and \textit{Bfu}AI are compatable with \textit{Nco}I.}
	\label{tab:pIND4_primers}
\end{sidewaystable}

