Sunteți pe pagina 1din 35

# Student Manual

pGLO Transformation
Lesson 1 Introduction to Transformation
!"#\$%&'#()*#+,-#.&((#/012,13#)#/1,405-10#6",."#)'#70"0\$&4#\$1)"'2,13)\$&,"8#90303*01
\$%)\$#)#70"0#&'#)#/&040#,2#:;<#.%&4%#/1,=&50'#\$%0#&"'\$1-4\$&,"'#2,1#3)6&"7#>4,50'#2,1?#)
/1,\$0&"8#@%&'#/1,\$0&"#7&=0'#)"#,17)"&'3#)#/)1\$&4-()1#\$1)&\$8#A0"0\$&4#\$1)"'2,13)\$&,"#(&\$01)((+
30)"'#4%)"70#4)-'05#*+#70"0'B#)"5#&"=,(=0'#\$%0#&"'01\$&,"#,2#)#70"0#&"\$,#)"#,17)"&'3#&"
,1501#\$,#4%)"70#\$%0#,17)"&'3C'#\$1)&\$8#A0"0\$&4#\$1)"'2,13)\$&,"#&'#-'05#&"#3)"+#)10)'#,2
*&,\$04%",(,7+8#!"#)71&4-(\$-10B#70"0'#4,5&"7#2,1#\$1)&\$'#'-4%#)'#21,'\$B#/0'\$B#,1#'/,&()70#
10'&'\$)"40#4)"#*0#70"0\$&4)((+#\$1)"'2,1305#&"\$,#/()"\$'8#!"#*&,10305&)\$&,"B#*)4\$01&)#4)"#*0#
70"0\$&4)((+#\$1)"'2,1305#.&\$%#70"0'#0")*(&"7#\$%03#\$,#5&70'\$#,&(#'/&(('8#!"#305&4&"0B#5&'0)'0'
4)-'05#*+#50204\$&=0#70"0'#)10#*07&""&"7#\$,#*0#\$10)\$05#*+#70"0#\$%01)/+D#\$%)\$#&'B#*+#70"0\$&4)((+
\$1)"'2,13&"7#)#'&46#/01',"C'#40(('#.&\$%#%0)(\$%+#4,/&0'#,2#\$%0#50204\$&=0#70"0#\$%)\$#4)-'0'#\$%0
5&'0)'08
E,-#.&((#-'0#)#/1,405-10#\$,#\$1)"'2,13#*)4\$01&)#.&\$%#)#70"0#\$%)\$#4,50'#2,1#A100"
F(-,10'40"\$#G1,\$0&"#>AFG?8#@%0#10)(H(&20#',-140#,2#\$%&'#70"0#&'#\$%0#*&,(-3&"0'40"\$#I0((+2&'%
!"#\$%&"'()*+,%&*'8#A100"#F(-,10'40"\$#G1,\$0&"#4)-'0'#\$%0#I0((+2&'%#\$,#2(-,10'40#)"5#7(,.#&"
\$%0#5)168#F,((,.&"7#\$%0#\$1)"'2,13)\$&,"#/1,405-10B#\$%0#*)4\$01&)#0J/10''#\$%0&1#"0.(+#)4K-&105
I0((+2&'%#70"0#)"5#/1,5-40#\$%0#2(-,10'40"\$#/1,\$0&"B#.%&4%#4)-'0'#\$%03#\$,#7(,.#)#*1&((&)"\$
7100"# 4,(,1#-"501#-(\$1)=&,(0\$#(&7%\$8
!"#\$%&'#)4\$&=&\$+B#+,-#.&((#(0)1"#)*,-\$#\$%0#/1,40''#,2#3,=&"7#70"0'#21,3#,"0#,17)"&'3#\$,
)",\$%01#.&\$%#\$%0#)&5#,2#)#/()'3&58#!"#)55&\$&,"#\$,#,"0#()170#4%1,3,',30B#*)4\$01&)#")\$-1)((+
4,"\$)&"#,"0#,1#3,10#'3)((#4&14-()1#/&040'#,2#:;<#4)((05#/()'3&5'8#G()'3&5#:;<#-'-)((+
4,"\$)&"'#70"0'#2,1#,"0#,1#3,10#\$1)&\$'#\$%)\$#3)+#*0#*0"02&4&)(#\$,#*)4\$01&)(#'-1=&=)(8#!"#")\$-10B
*)4\$01&)#4)"#\$1)"'201#/()'3&5'#*)46#)"5#2,1\$%#)((,.&"7#\$%03#\$,#'%)10#\$%0'0#*0"02&4&)(#70"0'8
@%&'#")\$-1)(#304%)"&'3#)((,.'#*)4\$01&)#\$,#)5)/\$#\$,#"0.#0"=&1,"30"\$'8#@%0#1040"\$#,44-1H
10"40#,2#*)4\$01&)(#10'&'\$)"40#\$,#)"\$&*&,\$&4'#&'#5-0#\$,#\$%0#\$1)"'3&''&,"#,2#/()'3&5'8
L&,H9)5C'#-"&K-0#/AMN#/()'3&5#0"4,50'#\$%0#70"0#2,1#AFG#)"5#)#70"0#2,1#10'&'\$)"40#\$,
\$%0#)"\$&*&,\$&4#)3/&4&((&"8#/AMN#)(',#&"4,1/,1)\$0'#)#'/04&)(#70"0#107-()\$&,"#'+'\$03B#.%&4%#4)"
*0#-'05#\$,#4,"\$1,(#0J/10''&,"#,2#\$%0#2(-,10'40"\$#/1,\$0&"#&"#\$1)"'2,1305#40(('8#@%0#70"0#2,1#AFG
4)"#*0#'.&\$4%05#,"#&"#\$1)"'2,1305 40(('#*+#)55&"7#\$%0#'-7)1#)1)*&",'0#\$,#\$%0#40(('C#"-\$1&0"\$
305&-38#O0(04\$&,"#2,1#40(('#\$%)\$#%)=0#*00"#\$1)"'2,1305#.&\$%#/AMN#:;<#&'#)44,3/(&'%05#*+
71,.\$%#,"#)"\$&*&,\$&4#/()\$0'8#@1)"'2,1305#40(('#.&((#)//0)1#.%&\$0#>.&(5H\$+/0#/%0",\$+/0?#,"
/()\$0'#",\$#4,"\$)&"&"7#)1)*&",'0B#)"5#2(-,10'40"\$#7100"#.%0"#)1)*&",'0#&'#&"4(-505#&"#\$%0#
"-\$1&0"\$#)7)1#305&-38#
E,-#.&((#*0#/1,=&505#.&\$%#\$%0#\$,,('#)"5#)#/1,\$,4,(#2,1#/012,13&"7#70"0\$&4#\$1)"'2,13)H
\$&,"8##
E,-1#\$)'6#.&((#*0P
Q8##@,#5,#\$%0#70"0\$&4#\$1)"'2,13)\$&,"8
R8##@,#50\$013&"0#\$%0#507100#,2#'-440''#&"#+,-1#022,1\$'#\$,#70"0\$&4)((+#)(\$01#)"#,17)"&'38#

28

## Lesson 1 Focus Questions

!"#\$#%&\$#%'&()%*+(,-.#\$&/-+(,%/"&/%(##.%/+%0#%/"+12"/%/"\$+12"%-(%/"#%3\$+*#,,%+4%
35&((-(2%&%,*-#(/-4-*%5&0+\$&/+\$)%-(6#,/-2&/-+(7%8#5+9%&\$#%&%4#9%4+\$%)+1%/+%3+(.#\$%&,%)+1
/&:#%+(%/"#%*"&55#(2#%+4%.+-(2%&%2#(#/-*%/\$&(,4+\$'&/-+(7%
;-(*#%,*-#(/-4-*%5&0+\$&/+\$)%-(6#,/-2&/-+(,%&\$#%.#,-2(#.%/+%2#/%-(4+\$'&/-+(%&0+1/%&
<1#,/-+(=%+1\$%4-\$,/%,/#3%'-2"/%0#%/+%4+\$'15&/#%&%<1#,/-+(%4+\$%/"-,%-(6#,/-2&/-+(7
Consideration 1: Can I Genetically Transform an Organism? Which
Organism?
>7%%!+%2#(#/-*&55)%/\$&(,4+\$'%&(%#(/-\$#%+\$2&(-,'=%)+1%'1,/%-(,#\$/%/"#%(#9%2#(#%-(/+%#6#\$)
*#55%-(%/"#%+\$2&(-,'7%?"-*"%+\$2&(-,'%-,%0#//#\$%,1-/#.%4+\$%/+/&5%2#(#/-*%/\$&(,4+\$'&/-+(@
+(#%*+'3+,#.%+4%'&()%*#55,=%+\$%+(#%*+'3+,#.%+4%&%,-(25#%*#55A

B7%%;*-#(/-,/,%+4/#(%9&(/%/+%:(+9%-4%/"#%2#(#/-*&55)%/\$&(,4+\$'#.%+\$2&(-,'%*&(%3&,,%-/,%(#9
/\$&-/,%+(%/+%-/,%+44,3\$-(2%&(.%41/1\$#%2#(#\$&/-+(,7%!+%2#/%/"-,%-(4+\$'&/-+(=%9"-*"%9+15.%0#
&%0#//#\$%*&(.-.&/#%4+\$%)+1\$%-(6#,/-2&/-+(=%&(%+\$2&(-,'%-(%9"-*"%#&*"%(#9%2#(#\$&/-+(%
.#6#5+3,%&(.%\$#3\$+.1*#,%<1-*:5)=%+\$%+(#%9"-*"%.+#,%/"-,%'+\$#%,5+95)A

C7%%;&4#/)%-,%&(+/"#\$%-'3+\$/&(/%*+(,-.#\$&/-+(%-(%*"++,-(2%&(%#D3#\$-'#(/&5%+\$2&(-,'7%?"&/
/\$&-/,%+\$%*"&\$&*/#\$-,/-*,%,"+15.%/"#%+\$2&(-,'%"&6#%E+\$%(+/%"&6#F%/+%0#%,1\$#%-/%9-55%(+/%"&\$'
)+1%+\$%/"#%#(6-\$+('#(/A

G7%%8&,#.%+(%/"#%&0+6#%*+(,-.#\$&/-+(,=%9"-*"%9+15.%0#%/"#%0#,/%*"+-*#%4+\$%&%2#(#/-*%
/\$&(,4+\$'&/-+(H%&%0&*/#\$-1'=%#&\$/"9+\$'=%4-,"=%+\$%'+1,#A%I#,*\$-0#%)+1\$%\$#&,+(-(27%

29

## Consideration 2: How Can I Tell if Cells Have Been Genetically

Transformed?
!"#\$%%&'(\$'&'("&)*\$%&*+&)","'-#&'.\$,/+*.0\$'-*,&-/&'*&#(\$,)"&\$,&*.)\$,-/01/&'.\$-'/
23(",*'43"56&7"+*."&\$,4&#(\$,)"&-,&'("&3(",*'43"&*+&\$,&*.)\$,-/0&#\$,&8"&9"'"#'"9:&\$
'(*.*;)(&"<\$0-,\$'-*,&*+&-'/&,\$';.\$%&23."='.\$,/+*.0\$'-*,5&3(",*'43"&0;/'&8"&0\$9"6
>**?&\$'&'("&#*%*,-"/&*+&!"#\$%&'#*,&4*;.&/'\$.'".&3%\$'"/6&>-/'&\$%%&*8/".@\$8%"&'.\$-'/&*.&
#(\$.\$#'".-/'-#/&'(\$'&#\$,&8"&9"/#.-8"9A
B("&+*%%*C-,)&3."='.\$,/+*.0\$'-*,&*8/".@\$'-*,/&*+&!"#\$%&' 0-)('&3.*@-9"&8\$/"%-,"&9\$'\$&'*
0\$?"&."+".",#"&'*&C(",&\$''"03'-,)&'*&9"'".0-,"&-+&\$,4&)","'-#&'.\$,/+*.0\$'-*,&(\$/
*##;.."96
\$5 D;08".&*+&#*%*,-"/
85 E-F"&*+&A

G5&'("&%\$.)"/'&#*%*,4
H5&'("&/0\$%%"/'&#*%*,4
I5&'("&0\$J*.-'4&*+&#*%*,-"/

#5 K*%*.&*+&'("&#*%*,-"/
95 L-/'.-8;'-*,&*+&'("&#*%*,-"/&*,&'("&3%\$'"
"5 M-/-8%"&\$33"\$.\$,#"&C(",&@-"C"9&C-'(&;%'.\$@-*%"'&2NM5&%-)('
+5 B("&\$8-%-'4&*+&'("&#"%%/&'*&%-@"&\$,9&."3.*9;#"&-,&'("&3."/",#"&*+&\$,&\$,'-8-*'-#&/;#(&\$/
\$03-#-%%-,
G. L"/#.-8"&(*C&4*;&#*;%9&;/"&'C*&>7O\$)\$.&3%\$'"/:&/*0"&!"#\$%&' \$,9&/*0"&\$03-#-%%-,&'*
9"'".0-,"&(*C&!"#\$%&' #"%%/&\$."&\$++"#'"9&84&\$03-#-%%-,6&&

H6 P(\$'&C*;%9&4*;&"<3"#'&4*;.&"<3".-0",'\$%&."/;%'/&'*&-,9-#\$'"&\$8*;'&'("&"++"#'&*+
\$03-#-%%-,&*,&'("&!"#\$%&' #"%%/Q

30

## Consideration 3: The Genes

!"#"\$%&'\$()#*+,(-)\$%,#'%#.,/."*'\$0"'%#*"(\$%,#',+'*,-"'#"1'234'%#\$,'\$0"'!"#\$%&'
&"//*5'6#')77%\$%,#'\$,',#"'/)(8"'&0(,-,*,-"9':)&\$"(%)',+\$"#'&,#\$)%#',#"',('-,("'*-)//
&%(&;/)('<%"&"*',+'234'&)//"7'</)*-%7*5'=/)*-%7'234';*;)//>'&,#\$)%#*'8"#"*'+,('-,("
\$0)#',#"'\$()%\$5'?&%"#\$%*\$*'&)#';*"')'<(,&"**'&)//"7'8"#"\$%&'"#8%#""(%#8'\$,'%#*"(\$'8"#"*
&,7%#8'+,('#"1'\$()%\$*'%#\$,')'</)*-%75'6#'\$0%*'&)*"9'\$0"'<!@A'</)*-%7'&)((%"*'\$0"'!B=
8"#"'\$0)\$'&,7"*'+,('\$0"'8(""#'+/;,("*&"#\$'<(,\$"%#')#7')'8"#"'C(&)D'\$0)\$'&,7"*'+,(')'
<(,\$"%#'\$0)\$'8%."*'\$0"':)&\$"(%)'("*%*\$)#&"'\$,')#')#\$%:%,\$%&5'E0"'8"#"\$%&)//>'"#8%#""("7
</)*-%7'&)#'\$0"#':"';*"7'\$,'8"#"\$%&)//>'\$()#*+,(-':)&\$"(%)'\$,'8%."'\$0"-'\$0%*'#"1'\$()%\$5

## pGLO plasmid DNA

GFP
Cell
wall

Flagellum

Bacterial
chromosomal
DNA

Pore

Beta-lactamase
(antibiotic resistance)

## Consideration 4: The Act of Transformation

E0%*'\$()#*+,(-)\$%,#'<(,&"7;("'%#.,/."*'\$0(""'-)%#'*\$"<*5'E0"*"'*\$"<*')("'%#\$"#7"7'\$,
%#\$(,7;&"'\$0"'</)*-%7'234'%#\$,'\$0"'!"#\$%&' &"//*')#7'<(,.%7"')#'"#.%(,#-"#\$'+,(''\$0"'&"//*
\$,'"F<("**'\$0"%('#"1/>')&G;%("7'8"#"*5
!"#\$"%&#'(&#)*+,#)-./\$01#234#'(5"67(#'(&#8&--#\$&\$95.:&#;"6#<0--=
H5 I*"')'\$()#*+,(-)\$%,#'*,/;\$%,#',+'J)J/K C&)/&%;-'&0/,(%7"D
K5 J)((>',;\$')'<(,&"7;("'("+"(("7'\$,')*'0")\$'*0,&L
>"5#'5.:/?"5\$&1#8&--/#'"#75"<#0:#'(&#)5&/&:8&#"?#.\$)080--0:#;"6#\$6/'=
M5 =(,.%7"'\$0"-'1%\$0'#;\$(%"#\$*')#7')'*0,(\$'%#&;:)\$%,#'<"(%,7'\$,':"8%#'"F<("**%#8'\$0"%(
#"1/>')&G;%("7'8"#"*

31

## Lesson 2 Transformation Laboratory

!) List
Workstation Check (!
!"#\$%&"\$'()*)+",- !"#\$%&"'()"*+)(,--'&\$()#."#)(./,'+)0\$)-%\$(\$*#)"#)1/,%)2/%3(#"#&/*)-%&/%
#/)0\$4&**&*4)#.&()'"0)"%\$)'&(#\$+)0\$'/25
.)#/0,)%&"\$'()*)+",(
!"#\$%&'#(#"%#\$%)-'"#\$
7/,%\$+)"4"%)-'"#\$()86)9:;)<)9:=">-;)6)9:=">-="%"?
A%"*(B/%>"#&/*)(/',#&/*
9:)*,#%&\$*#)0%/#.
C*/D,'"#&/*)'//-()
7&-\$#(
H/">)>&D%/#,0\$)./'+\$%=B'/"#
I/*#"&*\$%)B,'')/B)D%,(.\$+)&D\$)8B/">)D,-?
!"%3&*4)-\$*
I/-1)/B)J,&D3)K,&+\$

1#230\$%\$04#+\$0/
6
@
6
6
E)86)-3)/B)6F?
G
6
6
6
6

!6
5!
"
"
"
"
"
"
"
"
"
"

7,()\$#8)"\$9(%58"22",6%&"\$'()*)+",5)L)'&(#)/B)>"#\$%&"'(;)(,--'&\$()"*+)\$M,&->\$*#)#."#
(./,'+)0\$)-%\$(\$*#)"#)")D/>>/*)'/D"#&/*)#/)0\$)"DD\$((\$+)01)1/,%)#\$">)&()"'(/)'&(#\$+)0\$'/25
N\$.1+%"#\$+)-K9O)-'"(>&+
@<QI)2"#\$%)0"#.)"*+)#.\$%>/>\$#\$%
REQI)&*D,0"#/%
8/-#&/*"';)(\$\$)K\$*\$%"')9"0/%"#/%1 S3&''(TC*D,0"#&/*?

32

6)P&"'
6

"
"

"

Transformation Procedure

-pGLO

+pGLO

## !" #\$%&'()*&(+'),&-(./+0)(1&,1(12%&(!"#\$% \$*-(\$*)13&0(&"#\$%"(#\$%&'(%)13(12%&,(4/13

5)20(60)278,(*\$.&"(9'\$+&(13&.(/*(13&(:)\$.(12%&(0\$+;"

<" =7&*(13&(12%&,(\$*->(2,/*6(\$(,1&0/'&(10\$*,:&0(7/7&1>(10\$*,:&0(<?@(A'():(10\$*,:)0.\$1/)*(
,)'21/)*(BC\$C'<D(/*1)(&\$+3(12%&"

Transformation Solution

33

-pGLO

+pGLO

250 l

!" #\$%&'()*'()+,'-(./(0&'"(

Ice

+pGLO

1" 2-'(%(-)'30\$'(\$..4().(40&5(+4(!"#\$%&'(")*'*%+"*,"-!).(/\$!"63.7(8.+3(-)%3)'3(4\$%)'"(#0&5(+4
)*'(01234 )+,'(%/9(077'3-'()*'(\$..4(0/).()*'()3%/-6.37%)0./(-.\$+)0./(%)()*'(,.)).7(.6
)*'()+,'"(:40/()*'(\$..4(,');''/(8.+3(0/9'<(60/='3(%/9()*+7,(+/)0\$()*'('/)03'(&.\$./8(090-4'3-'9(0/()*'()3%/-6.37%)0./(-.\$+)0./(>;0)*(/.(6\$.%)0/=(&*+/5-?"(#\$%&'()*'()+,'(,%&5(0/
)*'()+,'(3%&5(0/()*'(0&'"(2-0/=(%(/';(-)'30\$'(\$..4@(3'4'%)(6.3()*'(51234 )+,'"((

(+pGLO)

(-pGLO)

+pGLO

A" B<%70/'()*'(4CDE(FGH(-.\$+)0./(;0)*()*'(2I(\$%74"(G.)'(8.+3(.,-'3J%)0./-"
K77'3-'(%(%(6"#.(/\$'("'**1 0/).()*'(4CDE(4\$%-709(FGH(-).&5()+,'"(L0)*93%;(%
\$..46+\$"(M*'3'(-*.+\$9(,'(%(60\$7(.6(4\$%-709(-.\$+)0./(%&3.--()*'(30/="(M*0-(0-(-070\$%3().
-''0/=(%(-.%48(60\$7(%&3.--(%(30/=(6.3(,\$.;0/=(-.%4(,+,,\$'-"(N0<()*'(\$..46+\$(0/).()*'
&'\$\$(-+-4'/-0./(.6()*'(01234 )+,'"(O\$.-'()*'()+,'(%/9(3')+3/(0)().()*'(3%&5(./(0&'"
H\$-.(&\$.-'()*'(51234 )+,'"(7*"%*. %99(4\$%-709(FGH().()*'(51234 )+,'"(L*8(/.)P(

## pGLO Plasmid DNA

(+pGLO)

34

(-pGLO)

!" #\$%&'()*+),*+)&'*-+.\$+/%*+0.1+23+4/\$&)*-"+5(6*+-&1*+).+7&-,+),*+)&'*-+(88+),*+9(:+;.9\$
/\$+),*+1(%6+-.+),*+'.)).4+.0+),*+)&'*-+-)/%6+.&)+(\$;+4(6*+%.\$)(%)+9/),+),*+/%*"

Rack

Ice

<" =,/8*+),*+)&'*-+(1*+-/))/\$>+.\$+/%*?+8('*8+:.&1+0.&1+@A+\$&)1/*\$)+(>(1+78()*-+.\$+),*+'.)).4
B\$.)+),*+8/;C+(-+0.88.9-D
E

@('*8+.\$*+!"#\$%& 78()*D

'(&)!*

@('*8+),*+!"#\$%&#\$+\$ 78()*D

'(&)!*

## @('*8+),*+.),*1+!"#\$%& 78()*D ,(&)!*

@('*8+),*+!" 78()*D

pGLO

,(&)!*

pGLO

LB/
amp/ara

LB/amp

pGLO

pGLO

LB/amp

LB

## F" -.\$/(012345 G-/\$>+),*+0.(4+1(%6+(-+(+,.8;*1?+)1(\$-0*1+'.),+),*+BHC+7I@J+(\$;+BKC

7I@J+)&'*-+/\$).+),*+9()*1+'(),?+-*)+()+LM.N?+0.1+*O(%)8:+P3+-*%.\$;-"+5(6*+-&1*+).
7&-,+),*+)&'*-+(88+),*+9(:+;.9\$+/\$+),*+1(%6+-.+),*+'.)).4+.0+),*+)&'*-+-)/%6+.&)+(\$;
4(6*+%.\$)(%)+9/),+),*+9(14+9()*1"
=,*\$+),*+P3+-*%.\$;-+(1*+;.\$*?+78(%*+'.),+)&'*-+'(%6+.\$+/%*"+Q.1+),*+'*-)+)1(\$-0.14()/.\$
1*-&8)-?+),*+)1(\$-0*1+01.4+),*+/%*+B3RNC+).+LMRN+(\$;+),*\$+'(%6+).+),*+/%*+4&-)+'*+1(7/;"
#\$%&'()*+)&'*-+.\$+/%*+0.1+M+4/\$&)*-"

Water bath
Ice

## 42C for 50 seconds

35

Ice

!" #\$%&'\$()*\$(+,-.(-&/),0/0/1()*\$()23\$4(5+&%()*\$(0-\$(,/6(78,-\$(&/()*\$(3\$/-*()&7"(97\$/
,()23\$(,/6:(240/1(,(/\$;(4)\$+08\$(707\$):(,66(<=>(?8(&5(@A(/2)+0\$/)(3+&)*()&()*\$()23\$(,/6
+\$-8&4\$(0)"(#\$7\$,)(;0)*(,(/\$;(4)\$+08\$(707\$)(5&+()*\$(&)*\$+()23\$"(B/-23,)\$()*\$()23\$4(5&+
C>(%0/2)\$4(,)(+&&%()\$%7\$+,)2+\$"(

-pGLO

+pGLO

250 l

LB broth

-pGLO

+pGLO

C>" D,7()*\$(-8&4\$6()23\$4(;0)*(E&2+(50/1\$+()&(%0F"(G40/1(,(/\$;(4)\$+08\$(707\$)(5&+(\$,-*()23\$:
707\$)(C>>(?8(&5()*\$()+,/45&+%,)0&/(,/6(-&/)+&8(4247\$/40&/4(&/)&()*\$(,77+&7+0,)\$(/2)+0\$/)
,1,+(78,)\$4"(

100 l

amp

+p

GL

LB
/amp/ara

-p

GL

LB/

GL

LB/

-p

amp

36

GL
LB

Control plates

Transformation plates

+p

!!" !"#\$%\$&#'\$"(#)*+#\$+,,-\$.,)\$#%/0\$-+%(#"#\$%&'()#*+'#,-,%'.,/0.,#'1'.23#(&0-.)#*+'#,-&4(5'
04#*+'#67#.-*&/'.*#(8(&#93#:-/5;23#,;(*/.8#*+'#42(*#,-&4(5'#04#(#.'<#,*'&/2'#200%#9(5;#(.)
40&*+#(5&0,,#*+'#%2(*'#,-&4(5'"#=>#?>@#ABC\$\$#@>>#=CCA#D?@>#@EC#FGFB"

+p

amp

GL

LB
/amp/ara

-p

GL

LB/

GL

LB/

amp

-p

+p

GL
LB

!H" \$*(5;#-%#30-&#%2(*',#(.)#*(%'#*+'I#*08'*+'&"#A-*#30-&#8&0-%#.(I'#(.)#52(,,#%'&/0)
0.#*+'#90**0I#04#*+'#,*(5;#(.)#%2(5'#*+'#,*(5;#04#%2(*',#1-"*2#\$2,'& /.#*+'#JKLM
/.5-9(*0&#-.*/2#*+'#.'N*#)(3"#

37

## Lesson 2 Review Questions

Name ___________________

!"#\$%"&'\$(("')*+,&-.).&.+-&.+.(/0*+,&/\$1%&%"21()2&.+23"%&)4"&#\$((\$3*+,&51"2)*\$+26
76 8+&34*'4&\$#&)4"&9(.)"2&3\$1(-&/\$1&":9"')&)\$&#*+-&;.')"%*.&<\$2)&(*="&)4"&\$%*,*+.(&
+\$+>)%.+2#\$%<"-&!"#\$%&' '\$(\$+*"2&/\$1&*+*)*.((/&\$;2"%?"-@&A:9(.*+&/\$1%&9%"-*')*\$+26

B6 C#&)4"%"&.%"&.+/&,"+")*'.((/&)%.+2#\$%<"-&;.')"%*.(&'"((2D&\$+&34*'4&9(.)"E2F&3\$1(-&)4"/
<\$2)&(*="(/&;"&(\$'.)"-@&A:9(.*+&/\$1%&9%"-*')*\$+26

G6 H4*'4&9(.)"2&24\$1(-&;"&'\$<9.%"-&)\$&-")"%<*+"&*#&.+/&,"+")*'&)%.+2#\$%<.)*\$+&4.2
\$''1%%"-@&H4/@

I6 H4.)&*2&<".+)&;/&.&'\$+)%\$(&9(.)"@&H4.)&91%9\$2"&-\$"2&.&'\$+)%\$(&2"%?"@

38

## Lesson 3 Data Collection and Analysis

A. Data Collection
!"#\$%&\$'()\$'%\$#*+(#',-*'-"(./0\$1'2%-3'()\$'(%.0#2-%3.(/-0'+."'*01\$%'0-%3.+'%--3'+/4)(/045
6)\$0'(*%0'-*('()\$'+/4)(#'.01')-+1'()\$'*+(%.&/-+\$('+/4)('-&\$%'()\$'7+.(\$#5'
85 9.%\$2*++,'-"#\$%&\$'.01'1%.:':).(',-*'#\$\$'-0'\$.;)'-2'()\$'2-*%'7+.(\$#5'<*(',-*%'1%.:/04#
/0'()\$'1.(.'(."+\$'/0'()\$';-+*30'-0'()\$'%/4)(5'=\$;-%1',-*%'1.(.'(-'.++-:',-*'(-';-37.%\$
-"#\$%&.(/-0#'-2'()\$'>!"#\$%&?';\$++#':/()',-*%'-"#\$%&.(/-0#'2-%'()\$'0-0@(%.0#2-%3\$1'
!"#\$%&'5'A%/(\$'1-:0'()\$'2-++-:/04'-"#\$%&.(/-0#'2-%'\$.;)'7+.(\$5
B5''C-:'3*;)'".;(\$%/.+'4%-:()'1-',-*'#\$\$'-0'\$.;)'7+.(\$D'%\$+.(/&\$+,'#7\$.E/04F''
G5''A).(';-+-%'.%\$'()\$'".;(\$%/.F''
H5''C-:'3.0,'".;(\$%/.+';-+-0/\$#'.%\$'-0'\$.;)'7+.(\$'I;-*0('()\$'#7-(#',-*'#\$\$J5'

&'()*+,-./0(

3*,0(4/*5,-./0"#2,-)(

K7LM!
MNO.37

K7LM!
MNO.37O.%.

&'()*+,-./0(

1/0-*/2"#2,-)(

@7LM!
MNO.37

@7LM!
MN

39

B. Analysis of Results
!"#\$%&'(\$&)\$*'+'\$','(-./.\$)&0\$+"/.\$/,1#.+/%'+/&,\$/.\$+&\$*#+#02/,#\$/)\$%#,#+/3\$+0',.)&02'+/&,
"'.\$&33400#*5
65 7"/3"\$&)\$+"#\$+0'/+.\$+"'+\$-&4\$&0/%/,'((-\$&8.#01#*\$)&0\$!"#\$%&' */*\$,&+\$.##2\$+&\$8#3&2#
'(+#0#*9\$\$:,\$+"#\$.;'3#\$8#(&<\$(/.+\$+"#.#\$4,+0',.)&02#*\$+0'/+.\$',*\$"&<\$-&4\$'00/1#*\$'+\$+"/.
','(-./.\$)&0\$#'3"\$+0'/+\$(/.+#*5
!"#\$#%&'()"&#)

*%&'+,#,(-.(-/,0"1&)#-%,

=5 >)\$+"#\$!"#\$%&' +0'/+.\$-&4\$&0/%/,'((-\$,&+#*?\$<"/3"\$.##2\$,&<\$+&\$8#\$./%,/)/3',+(-\$*/))#0#,+
')+#0\$;#0)&02/,%\$+"#\$+0',.)&02'+/&,\$;0&3#*40#9\$@/.+\$+"&.#\$+0'/+.\$8#(&<\$',*\$*#.30/8#\$+"#
3"',%#.\$+"'+\$-&4\$&8.#01#*5
203()"&#)

!/,0"104(56&%\$0

A5 :)\$+"#\$%#,#+/3'((-\$+0',.)&02#*\$3#((.\$"'1#\$'3B4/0#*\$+"#\$'8/(/+-\$+&\$(/1#\$/,\$+"#\$;0#.#,3#\$&)\$+"#
',+/8/&+/3\$'2;/3/((/,?\$+"#,\$<"'+\$2/%"+\$8#\$/,)#00#*\$'8&4+\$+"#\$&+"#0\$%#,#.\$&,\$+"#\$;('.2/*
+"'+\$-&4\$4.#*\$/,\$-&40\$+0',.)&02'+/&,\$;0&3#*40#9\$

C5 D0&2\$+"#\$0#.4(+.\$+"'+\$-&4\$&8+'/,#*?\$"&<\$3&4(*\$-&4\$;0&1#\$+"'+\$+"#\$3"',%#.\$+"'+\$&33400#*
<#0#\$*4#\$+&\$+"#\$;0&3#*40#\$+"'+\$-&4\$;#0)&02#*9

40

## Lesson 3 Review Questions

Name _____________________

Whats Glowing?
!"#\$#"%&'()*+),-#.()),#+'%'(#/*#'0*)(1)2#/,#-3)#!"#\$%&' +'%',/)*#-3),#\$#,)4#5&)*-/',
6/.3-#4)%%#0)#(\$/*)27#893\$-#\$()#-3)#-4'#:'**/0%)#*'&(+)*#'"#"%&'()*+),+)#4/-3/,#-3)
+'%',/)*#43),#);:'*)2#-'#<=#%/.3->?
@;:%\$/,A
BC D)+\$%%#43\$-#E'&#'0*)(1)2#43),#E'&#*3/,)2#-3)#<=#%/.3-#',-'#\$#*\$6:%)#'"#'(/./,\$%
:FGH#:%\$*6/2#IJK#\$,2#2)*+(/0)#E'&(#'0*)(1\$-/',*C

LC 93/+3#'"#-3)#-4'#:'**/0%)#*'&(+)*#'"#-3)#"%&'()*+),+)#+\$,#,'4#0)#)%/6/,\$-)2>

MC 93\$-#2')*#-3/*#'0*)(1\$-/',#/,2/+\$-)#\$0'&-#-3)#*'&(+)#'"#-3)#"%&'()*+),+)>##

NC I)*+(/0)#-3)#)1/2),+)#-3\$-#/,2/+\$-)*#43)-3)(#E'&(#\$--)6:-#\$-#:)("'(6/,.#\$#.),)-/+
-(\$,*"'(6\$-/',#4\$*#*&++)**"&%#'(#,'-#*&++)**"&%C

41

## Lesson 3 Review Questions

Name ____________________

## The Interaction between Genes and Environment

!""#\$%&%'(\$%)\$*"+,\$-"+,\$./%)012\$3"\$*"+\$"410,50\$1"60\$!"#\$%&' &,"7'(&\$"(\$)80\$!9\$./%)0\$)8%)
:"01\$(")\$;"()%'(\$%6.';'//'(\$",\$%,%4'("10<

=2 >,"6\$*"+,\$,01+/)1?\$;%(\$*"+\$)0//\$'-\$)8010\$4%;)0,'%\$%,0\$%6.';'//'(\$,01'1)%()\$4*\$/""#'(&\$%)
)806\$"(\$)80\$!9\$./%)0<\$\$@A./%'(\$*"+,\$%(170,2

B2 C"7\$7"+/:\$*"+\$;8%(&0\$)80\$4%;)0,'%D1\$0(5',"(60()E)80\$./%)0\$)80*\$%,0\$&,"7'(&\$"(E)"
401)\$)0//\$'-\$)80*\$%,0\$%6.';'//'(\$,01'1)%()<

F2 G0,*\$"-)0(\$%(\$",&%('16D1\$),%')1\$%,0\$;%+10:\$4*\$%\$;"64'(%)'"(\$"-\$')1\$&0(01\$%(:\$')1\$0(5',"(60()2
H8'(#\$%4"+)\$)80\$&,00(\$;"/",\$*"+\$1%7\$'(\$)80\$&0(0)';%//*\$),%(1-",60:\$4%;)0,'%I
%2

J8%)\$)7"\$-%;)",1\$6+1)\$40\$.,010()\$'(\$)80\$4%;)0,'%D1\$0(5',"(60()\$-",\$*"+\$)"\$100\$)80
&,00(\$;"/",<\$KC'()I\$\$"(0\$-%;)",\$'1\$'(\$)80\$./%)0\$%(:\$)80\$")80,\$-%;)",\$'1\$'(\$8"7\$*"+
/""#\$%)\$)80\$4%;)0,'%L2

42

J8%)\$:"\$*"+\$)8'(#\$0%;8\$"-\$)80\$)7"\$0(5',"(60()%/\$-%;)",1\$*"+\$/'1)0:\$%4"50\$%,0
:"'(&\$)"\$;%+10\$)80\$&0(0)';%//*\$),%(1-",60:\$4%;)0,'%\$)"\$)+,(\$&,00(<

;2

J8%)\$%:5%()%&0\$7"+/:\$)80,0\$40\$-",\$%(\$",&%('16\$)"\$40\$%4/0\$)"\$)+,(\$"(\$",\$"--\$.%,)';+/%,
&0(01\$'(\$,01."(10\$)"\$;0,)%'(\$;"(:')'"(1<

42

## Lesson 4 Extension Activity: Calculate Transformation

Efficiency
!"#\$%&'()%)*+,%-&%).-+%-&/'+)-0*)-"&%1-22%3'%)"%2'*\$&%."1%)"%4')'\$5-&'%).'%'()'&)%)"
1.-6.%7"#%0'&')-6*227%)\$*&+8"\$5'4%!"#\$%&' 6'22+9%:.-+%;#*&)-)*)-/'%5'*+#\$'5'&)%-+%\$'8'\$\$'4
)"%*+%).'%)\$*&+8"\$5*)-"&%'88-6-'&679%%
<&%5*&7%'(='\$-5'&)+>%-)%-+%-5="\$)*&)%)"%0'&')-6*227%)\$*&+8"\$5%*+%5*&7%6'22+%*+%="++-32'9%?"\$
'(*5=2'>%-&%+"5'%)7='+%"8%0'&'%).'\$*=7>%6'22+%*\$'%6"22'6)'4%8\$"5%).'%=*)-'&)>%)\$*&+8"\$5'4%-&%).'
2*3"\$*)"\$7>%*&4%).'&%=#)%3*6,%-&)"%).'%=*)-'&)9%:.'%5"\$'%6'22+%).*)%*\$'%)\$*&+8"\$5'4%)"%=\$"4#6'
).'%&''4'4%=\$")'-&>%).'%5"\$'%2-,'27%).*)%).'%).'\$*=7%1-22%1"\$,9%:.'%)\$*&+8"\$5*)-"&%'88-6-'&67%-+
6*26#2*)'4%)"%.'2=%+6-'&)-+)+%4')'\$5-&'%."1%1'22%).'%)\$*&+8"\$5*)-"&%-+%1"\$,-&09
!"#\$!%&'
!"#%*\$'%*3"#)%)"%6*26#2*)'%).'%)\$*&+8"\$5*)-"&%'88-6-'&67>%1.-6.%0-/'+%7"#%*&%-&4-6*)-"&
"8%."1%'88'6)-/'%7"#%1'\$'%-&%0'))-&0%@AB%5"2'6#2'+%-&)"%3*6)'\$-*2%6'22+9%:\$*&+8"\$5*)-"&
'88-6-'&67%-+%*%&#53'\$9%<)%\$'=\$'+'&)+%).'%)")*2%&#53'\$%"8%3*6)'\$-*2%6'22+%).*)%'(=\$'++%).'
0\$''&%=\$")'-&>%4-/-4'4%37%).'%*5"#&)%"8%@AB%#+'4%-&%).'%'(='\$-5'&)9%C<)%)'22+%#+%).'%)")*2
&#53'\$%"8%3*6)'\$-*2%6'22+%)\$*&+8"\$5'4%37%"&'%5-6\$"0\$*5%"8%@AB9D%:.'%)\$*&+8"\$5*)-"&%
'88-6-'&67%-+%6*26#2*)'4%#+-&0%).'%8"22"1-&0%8"\$5#2*E
:\$*&+8"\$5*)-"&%'88-6-'&67%F%%:")*2%&#53'\$%"8%6'22+%0\$"1-&0%"&%).'%*0*\$%=2*)'
B5"#&)%"8%@AB%+=\$'*4%"&%).'%*0*\$%=2*)'%C-&%G0D
:.'\$'8"\$'>%3'8"\$'%7"#%6*&%6*26#2*)'%).'%'88-6-'&67%"8%7"#\$%)\$*&+8"\$5*)-"&>%7"#%1-22
&''4%)1"%=-'6'+%"8%-&8"\$5*)-"&E
()* !"#\$+,+%-\$./01#2\$,3\$42##.\$3-/,2#&5#.+\$5,-,.6#&\$42,76.4\$,.\$8,/2\$9:;%0<;%2%
<-%+#=
(>* !"#\$+,+%-\$%0,/.+\$,3\$<?9@\$<-%&06A\$BCD\$6.\$+"#\$1%5+#26%-\$5#--&\$&<2#%A\$,.\$+"#
9:;%0<;%2%\$<-%+#=

43

## 1. Determining the Total Number of Green Fluorescent Cells

!"#\$%&'()*&+,-#./-#*#&/"#0%&1%#*&#&23&"45607&8#\$6&\$("(1'&(1&06%&/"#0%&\$#1&9%&#::).%;&0(
9%&;%*4<%;&=*(.&#&:415"%&\$%""7&>:&41;4<4;)#"&\$%"":&*%/*(;)\$%?&.(*%&#1;&.(*%&\$%"":&#*%
=(*.%;&#1;&;%<%"(/&410(&@6#0&4:&0%*.%;&#&\$("(1'7&A6%&.(:0&;4*%\$0&@#'&0(&;%0%*.41%&06%
!"!#\$%&'()*+ ",%-+**&%,\$'"+*./*&!%/*\$\$. 4:&0(&\$()10&06%&\$("(14%:&(1&06%&/"#0%7
810%*&06#0&1).9%*&6%*%&

A(0#"&1).9%*&(=&\$%"":&L
TTTTTTT

## 2. Determining the Amount of pGLO DNA in the Bacterial Cells Spread on

the LB/amp/ara Plate
B%&1%%;&0@(&/4%\$%:&(=&41=(*.#04(1&0(&=41;&()0&06%&#.()10&(=&/C+D&EF>&41&06%&9#\$0%*4#"
\$%"":&:/*%#;&(1&06%&+,-#./-#*#&/"#0%&41&064:&%G/%*4.%107&H#I&B6#0&@#:&06%&0(0#"&#.()10&(=
EF>&@%&9%5#1&06%&%G/%*4.%10&@406?&#1;&H9I&B6#0&=*#\$04(1&(=&06%&EF>&H41&06%&9#\$0%*4#I&
#\$0)#""'&5(0&:/*%#;&(10(&06%&+,-#./-#*#&/"#0%:7&
D1\$%&'()&\$#"\$)"#0%&064:&;#0#?&'()&@4""&1%%;&0(&.)"04/"'&06%&!"!#\$%#("'&!%",%0123%456
):%;&41&064:&%G/%*4.%10&9'&06%&,+#/!7"&%",%456 '()&:/*%#;&(1&06%&+,-#./-#*#&/"#0%7&A6%
#1:@%*&0(&064:&.)"04/"4\$#04(1&@4""&0%""&'()&06%&#.()10&(=&/C+D&EF>&41&06%&9#\$0%*4#"&\$%"":
06#0&@%*%&:/*%#;&(1&06%&+,-#./-#*#&/"#0%7
a. Determining the Total Amount of pGLO plasmid DNA
A6%&0(0#"&#.()10&(=&EF>&@%&9%5#1&@406&4:&%J)#"&0(&06%&/*(;)\$0&(=&06%&\$(1\$%10*#04(1&#1;
06%&0(0#"&<(").%&):%;?&(*
HEF>&41&K5I&&L&&H\$(1\$%10*#04(1&(=&EF>&41&K5-K"I&&G&&H<(").%&(=&EF>&41&K"I
M1&064:&%G/%*4.%10&'()&):%;&NO&K"&(=&/C+D&#0&\$(1\$%10*#04(1&(=&O7OP&K5-K"7&A64:&.%#1:&06#0
%#\$6&.4\$*("40%*&(=&:(")04(1&\$(10#41%;&O7OP&K5&(=&/C+D&EF>7&Q#"\$)"#0%&06%&!"!#\$%#("'&!
",%456%):%;&41&064:&%G/%*4.%107

8"!#\$%#("'&!%",%0123%456%9:-;
):%;&41&064:&%G/%*4.%10&L
TTTTTTTTTT

810%*&06#0&1).9%*&6%*%&

R(@&@4""&'()&):%&064:&/4%\$%&(=&41=(*.#04(1S

44

!" #\$%\$&'()()*+%,\$+-&./%(0)+0-+1234+15.6'(7+#89+:()+%,\$+!./%\$&(.;+%,.%+./%<.55=+*0%
61&\$.7+0)%0+%,\$+3>?.'1?.&.+15.%\$@ !"#\$%&#'(&)**&(+%&,-.&/'0&)11%1&('&(+%&2)\$(%3")*&\$%**4
5"**&2%&(3)#46%33%1&('&(+%&)7)3&8*)(%9&/'0&#%%1&('&6"#1&'0(&5+)(&63)\$("'#&'6&(+%&,-.&5)4&)\$(0)**/
483%)1&'#('&(+%&:;<)=8<)3)&8*)(%>&?'&1'&(+"49&1"@"1%&(+%&@'*0=%&'6&,-.&/'0&483%)1&'#&(+%
:;<)=8<)3)&8*)(%&2/&(+%&('()*&@'*0=%&'6&*"A0"1&"#&(+%&(%4(&(02%&\$'#()"#"#7&(+%&,-.>&.&6'3=0*)
6'3&(+"4&4()(%=%#(&"4
U3)\$("'#&'6&,-.&04%1&&S&& B'*0=%&483%)1&'#&:;<)=8&8*)(%&CD*E
?'()*&4)=8*%&@'*0=%&"#&(%4(&(02%&CD*E
F'0&483%)1&GHH&D*&'6&\$%**4&\$'#()"#"#7&,-.&63'=&)&(%4(&(02%&\$'#()"#"#7&)&('()*&@'*0=%&'6
IGH&D*&'6&4'*0("'#>&,'&/'0&3%=%=2%3&5+/&(+%3%&"4&IGH&D*&('()*&4'*0("'#J&:''K&"#&(+%&*)2'3)('3/
83'\$%103%&)#1&*'\$)(%&)**&(+%&4(%84&5+%3%&/'0&)11%1&*"A0"1&('&(+%&3%)\$("'#&(02%>&.11&(+%&@'*0=%4>
L4%&(+%&)2'@%&6'3=0*)&('&\$)*\$0*)(%&(+%&-&./%(0)+0-+1234+15.6'(7+#89 /'0&483%)1
'#&(+%&:;<)=8<)3)&8*)(%>

M#(%3&(+)(&#0=2%3&+%3%&&
N

U3)\$("'#&'6&,-.&S
VVVVVVVV

O'5&5"**&/'0&04%&(+"4&8"%\$%&'6&"#6'3=)("'#J

A0B+,0C+'.)=+'(/&0*&.'6+0-+1234+#89+7(7+=0<+61&\$.7+0)+%,\$+3>?.'1?.&.+15.%\$6D

?'&)#45%3&(+"4&A0%4("'#9&/'0&5"**&#%%1&('&=0*("8*/&(+%&%0%.5+.'0<)%+0-+1234+#89
<6\$7 "#&(+"4&%P8%3"=%#(&2/&(+%&-&./%(0)+0-+1234+#89 /'0&483%)1&'#&(+%&:;<)=8<)3)&8*)(%>
8Q:R&,-.&483%)1&"#&D7&&S&&?'()*&)='0#(&'6&,-.&04%1&"#&D7&&P&&63)\$("'#&'6&,-.&04%1

M#(%3&(+)(&#0=2%3&+%3%&
N

T+)(&5"**&(+"4&#0=2%3&(%**&/'0J

45

8Q:R&,-.&483%)1&CD7E&S
VVVVVVVV

!""#\$%&\$%''\$(")*\$+%'+)'%&,"-.\$%/"012\$31+,41\$56,+6\$"7\$&61\$-)8/1*.\$(")\$+%'+)'%&14
/1'"-9\$,-\$&61\$&%/'1\$/1'"52\$:,''\$,-\$&61\$7"''"5,-9\$&%/'12

;)8/1*\$"7\$+"'"-,1.\$"!TH%8<H%*%\$<'%&1\$@
U,+*"9*%8.\$"7\$<=!>\$3;A
.<*1%4\$"-\$&61\$<'%&1.
;"5\$).1\$&61\$4%&%\$,-\$&61\$&%/'1\$&"\$+%'+)'%&1\$&61\$177,+,1-+(\$"7\$&61\$<=!>\$&*%-.7"*8%&,"?*%-.7"*8%&,"-\$177,+,1-+(\$@\$\$?"&%'\$-)8/1*\$"7\$+1''.\$9*"5,-9\$"-\$&61\$%9%*\$<'%&1
A8")-&\$"7\$3;A\$.<*1%4\$"-\$&61\$%9%*\$<'%&1

?*%-.7"*8%&,"-\$177,+,1-+(\$@

B-&1*\$&6%&\$-)8/1*\$61*1\$

VVVVV\$&*%-.7"*8%-&.HI9

Analysis
?*%-.7"*8%&,"-\$177,+,1-+(\$+%'+)'%&,"-.\$*1.)'&\$,-\$01*(\$'%*91\$-)8/1*.2\$C+,1-&,.&.\$"7&1-\$).1
%\$8%&618%&,+%'\$.6"*&6%-4\$*171**14\$&"\$%.\$.+,1-&,7,+\$-"&%&,"-2\$:"*\$1D%8<'1E\$,7\$&61\$+%'+)'%&14
&*%-.7"*8%&,"-\$177,+,1-+(\$,.\$FEGGG\$/%+&1*,%HI9\$"7\$3;AE\$&61(\$"7&1-\$*1<"*&\$&6,.\$-)8/1*\$%.J
!"# \$%&'()*%+&'\$(,-.
N

KFGL ,.\$%-"&61*\$5%(\$"7\$.%(,-9\$FG\$D\$FG\$D\$FG\$"*\$FEGGGM

O"5\$5")'4\$.+,1-&,.&.\$*1<"*&\$FGEGGG\$&*%-.7"*8%-&.HI9\$,-\$.+,1-&,7,+\$-"&%&,"-P

Q%**(,-9\$&6,.\$,41%\$%\$',&&'1\$7%*&61*E\$.)<<".1\$.+,1-&,.&.\$+%'+)'%&14\$%-\$177,+,1-+(\$"7\$REGGG
/%+&1*,%HI9\$"7\$3;A2\$?6,.\$5")'4\$/1\$*1<"*&14\$%.J
/010!"# \$%&'()*%+&'\$(,-.
N

KR\$&,81.\$FEGGGM

O"5\$5")'4\$.+,1-&,.&.\$*1<"*&\$SGEGGG\$&*%-.7"*8%-&.HI9\$,-\$.+,1-&,7,+\$-"&%&,"-P

46

!"#\$%&"'(\$#)'*+(#,\$-%\$./011\$23'"4%53*'"24678\$9#3#\$:'(:;('2#</\$2=#"\$2=#\$4:&#"2&%&:\$"52'2&5"
%53\$2=&4\$";*>#3\$95;(<\$>#,
!"#\$%\$&'( )*+,-./*0+,)-123

?.@0\$2&*#4\$A/111B

C&*&('3(D,
E/011\$F\$E@0\$)\$A1G

.HA/111\$F\$\$.@HA\$)\$A1E

./I.1/111\$F\$.@I.\$)\$A10

K59\$95;(<\$4:&#"2&424\$3#+532\$L01/111\$23'"4%53*'"24678\$&"\$4:&#"2&%&:\$"52'2&5"M

N#+532\$D5;3\$:'(:;('2#<\$23'"4%53*'2&5"\$#%%&:&#":D\$&"\$4:&#"2&%&:\$"52'2&5"@

O4#\$'\$4#"2#":#\$53\$295\$25\$#)+('&"\$9='2\$D5;3\$:'(:;('2&5"\$5%\$23'"4%53*'2&5"\$#%%&:&#":D
*#'"4@

P&52#:="5(58&424\$'3#\$&"\$8#"#3'(\$'83##*#"2\$2='2\$2=#\$23'"4%53*'2&5"\$+3525:5(\$2='2\$D5;
='Q#\$R;42\$:5*+(#2#<\$8#"#3'((D\$='4\$'\$23'"4%53*'2&5"\$#%%&:&#":D\$5%\$>#29##"\$S@1\$)\$A1. '"<\$H@1
)\$A1G 23'"4%53*'"24\$+#3\$*&:3583'*\$5%\$TUV@
J

K59\$<5#4\$D5;3\$23'"4%53*'2&5"\$#%%&:&#":D\$:5*+'3#\$9&2=\$2=#\$'>5Q#M

-"\$2=#\$2'>(#\$>#(59/\$3#+532\$2=#\$23'"4%53*'2&5"\$#%%&:&#":D\$5%\$4#Q#3'(\$5%\$2=#\$2#'*4\$&"\$2=#
:('44@

45+0

6..7875,89

K59\$<5#4\$D5;3\$23'"4%53*'2&5"\$#%%&:&#":D\$:5*+'3#\$9&2=\$2=#&34M

47

"#\$%&\$#'()'*()'+#,-./+0#'1/,)(..1%1(,%2)/.)'*()./\$\$/31,4)(56(+10(,')&-1,4)'*()1,./+0#7
'1/,)#,8)'*()+(-&\$'-)\$1-'(8)9(\$/3:

!"#\$%&'()*+\$,-.,/.01'0*-.2\$3435\$6786&
9:3\$6&\$;';&9 01'.(<-1)'0*-.\$(-&=0*-.
>3\$6&\$%?@A\$%&'()*+\$(-&=0*-.
9:3\$6&\$@B\$C1-0D
>33\$6&\$,/&&(\$(%1/'+\$-.\$'7'1
99E\$,-&-.*/(\$-<\$01'.(<-1)'.0(
;1\$\$)1,)'*()./\$\$/31,4)%*#+')#,8)-*/3)2/&+)%#\$%&\$#'1/,-)'/)2/&+)'(#%*(+<
G&09(+)/.)%/\$/,1(-)/,)JKD#06D#+#)6\$#'()P
Q1%+/4+#0-)/.)FGH)-6+(#8)/,)'*()6\$#'(-)P
R+#,-./+0#'1/,)(..1%1(,%2)P

=5'+#)"+(81')"*#\$\$(,4(<

>.)#)6#+'1%&\$#+)(56(+10(,')3(+()?,/3,)'/)*#@()#)'+#,-./+0#'1/,)(..1%1(,%2)/.)A)5)BCA
9#%'(+1#DE4)/.)FGHI)*/3)0#,2)'+#,-./+0#,')%/\$/,1(-)3/&\$8)9()(56(%'(8)'/)4+/3)/,)'*(
JKD#06D#+#)6\$#'(L)M/&)%#,)#--&0()'*#')'*()%/,%(,'+#'1/,)/.)FGH)#,8).+#%'1/,)/.)%(\$\$-6+(#8)/,)'*()JK)#4#+)#+()'*()-#0()#-)'*#')/.)'*()6NJO)\$#9/+#'/+2:

48

## Appendix A Historical Links to Biotechnology

!"#\$#%"&'\$()*'+,-#*.')"#+(/',(/'0('+("+)1*1,)"+%(/",)#*23(4+(56789(:*101*"&;(<*"--")/9('
=#+0#+(>/2,"&"'+(?#*;"+%("+('(>')/#\$#%2(\$'@#*')#*29(&#+0A&)10('+(1B>1*".1+)()/')(/1
?#A\$0(+1C1*(@1('@\$1()#(-A\$\$2("+)1*>*1)(',(\$#+%(',(/1(\$"C103(<*"--")/(>1*.'+1+)\$2(&/'+%10
D)*'+,-#*.10E('(,'-19(+#+>')/#%1+"&(@'&)1*"'\$(,)*'"+(#-(>+1A.#&#&&A,("+)#('(01'0\$2
>')/#%1+"&(,)*'"+3(F1('&&#.>\$",/10()/",('.'G"+%(&/'+%1("+()/1(@'&)1*"'(@2()*1')"+%()/1(,'-1
@'&)1*"'(?")/(/1')H;"\$\$10(01'0\$2(@'&)1*"'3(4+()/",(."B)A*1(#-()/1()?#(@'&)1*"'\$(,)*'"+,()/1*1
?1*1(+#(\$"C"+%9(C"*A\$1+)(@'&)1*"'9(@A)()/1(."B)A*1(;"\$\$10()/1(."&1(")(?',("+I1&)10("+)#3(F1
*1>1')10()/1(1B>1*".1+)(.'+2()".1,9('\$?'2,(?")/()/1(,'.1(*1,A\$),3(F1('+0(.'+2(#-(/",(
&#\$\$1'%A1,(?1*1(C1*2(>1*>\$1B103(J/')()*'+,-#*.10(,'-1(@'&)1*"'("+)#()/1(01'0\$2(;"\$\$1*,K
L'+2(21'*,(\$')1*9()/",(?#A\$0(&#.1()#(@1(;+#?+(',()/1(-"*,)(*1&#*010(&',1(#-(@"#\$#%"&'\$
)*'+,-#*.')"#+(&#+0A&)10("+('(\$'@#*')#*29('+0(+#(#+1(&#A\$0(1B>\$'"+(")3(<*"--")/(0"0(+#)
;+#?(#-(MNO9(@A)(;+1?()/1()*'+,-#*.')"#+(?',("+/1*")'@\$13(O,('+2(,"+%\$1(>#"+)("+(/",)#*2
&'+(@19(<*"--")/P,(1B>1*".1+),("+()*'+,-#*.')"#+(&'+(@1(,11+(',()/1(@"*)/(#-('+'\$2)"&'\$(
%1+1)"&(.'+">A\$')"#+()/')(/',(\$10()#(*1&#.@"+'+)(MNO('+0(@"#)1&/+#\$#%29('+0()/1
>*#,>1&),(-#*(/A.'+(%1+1(.'+">A\$')"#+3((
4+(56QQ9(,"B)11+(21'*,('-)1*(<*"--")/P,(1B>1*".1+)9('(*1,1'*&/(%*#A>(')(R#&;1-1\$\$1*
4+,)")A)19(\$10(@2(S,?'\$0(T3(OC1*29(>A@\$",/10('(>'>1*()/')(&'.1(0"*1&)\$2(-*#.()/1(?#*;(#<*"--")/3(UJ/')(",()/1(,A@,)'+&1(*1,>#+,"@\$1KV(OC1*2(?#A\$0(',;(/",(&#?#*;1*,3(J#*;"+%
?")/()/1(,'.1(,)*'"+,(#-(>+1A.#+"'H&'A,"+%(@'&)1*"'9(OC1*2('+0(/",(&#?#*;1*,(>*#C"010('
*"%#*#A,('+,?1*()#()/')(WA1,)"#+3(T/12(>*#C10()/')()/1(,A@,)'+&1(",(MNO9('+0()/')(@"#\$#%"&'\$
)*'+,-#*.')"#+(",(>*#0A&10(?/1+(&1\$\$,()';1(A>('+0(1B>*1,,(-#*1"%+(MNO3(O\$)/#A%/(")()##;
.'+2(21'*,(-#*(&*10")()#(@1(%"C1+()#(OC1*29()#0'2(/1(",(A+"C1*,'\$\$2('&;+#?\$10%10(-#*()/",
-A+0'.1+)'\$('0C'+&1("+(@"#\$#%"&'\$(;+#?\$10%13(!A"\$0"+%(A>#+()/1(?#*;(#-(OC1*2('+0(
#)/1*,9(M#A%\$',(F'+'/'+(01C1\$#>10()/1()1&/+"WA1(#-(&#\$#+2()*'+,-#*.')"#+(A,10("+()/",
"+C1,)"%')"#+359(7
Historical Context
!"#"\$%&'()*#+,-).*\$%-#
58XYZ<*1%#*([#/'++(L1+01\$\(L1+01\$(>*1,1+)10(/",(-"+0"+%,(01,&*"@"+%()/1(>*"+&">\$1,(@2
?/"&/(%1+1)"&()*'"),('*1(>',,10(-*#.(>'*1+)()#(#--,>*"+%3(:*#.(/",(?#*;()/1(&#+&1>)(#)/1(%1+1(',()/1(@',"&(A+")(#-(/1*10")2(?',(01*"C103((
56]]Z^'*\$(^#**1+,9(FA%#(M1(_*"1,9(`*"&/(T,&/1*.';\(a\$'+)(%1+1)"&",),(&#+0A&)"+%(
"+/1*")'+&1(,)A0"1,(A+&#C1*10()/')()/1"*(?#*;(?',(1,,1+)"'\$\$2('(0A>\$"&')1(#-(?#*;(
>1*-#*.10(+1'*\$2(-#A*(01&'01,(1'*\$"1*(@2('+(A+;+#?+(OA,)*"'+(OA%A,)"+"'+(.#+;9
<*1%#*([#/'++(L1+01\$9(?/#(,)A0"10(>1',3
5678Z:*101*"&;(<*"--")/\(<*"--")/()*'+,-#*.10(+#+>')/#%1+"&(/%01-&-&&2+'0#"2.-#%* "+)#
>')/#%1+"&(@'&)1*"'(A,"+%(/1')H;"\$\$10(C"*A\$1+)(@'&)1*"'3(F1(,A%%1,)10()/')()/1()*'+,-#*."+%
-'&)#*(/'0(,#.1)/"+%()#(0#(?")/()/1(>#\$2,'&&/'*"01(&'>,A\$1(,2+)/1,",3(<*"--")/(0"0(+#)
;+#?(#-(MNO9(@A)(;+1?()/1()*'+,-#*.')"#+(?',("+/1*")'@\$13(<*"--")/P,(1B>1*".1+),("+
)*'+,-#*.')"#+(&'+(@1(,11+(',()/1(@"*)/(#-('+'\$2)"&'\$(%1+1)"&(.'+">A\$')"#+()/')(/',(\$10()#
*1&#.@"+'+)(MNO()1&/+#\$#%2('+0()/1(>*#,>1&),(-#*(/A.'+(%1+1(.'+">A\$')"#+3
56QQZS,?'\$0(OC1*29(^#\$"+(L'&=1#0\((OC1*2('+0(/",(&#\$\$1'%A1,('++#A+&10()/')()/12(/'0
",#\$')10()/1()*'+,-#*."+%(-'&)#*()#('(/"%/(>A*")29('+0(")(?',(MNO3(b"+&1()/",(&\$',,"&(
1B>1*".1+)("+(.#\$1&A\$'*(%1+1)"&,9()*'+,-#*.')"#+9(&#+IA%')"#+(D@'&)1*"'\$(.')"+%E9('+0
)*'+,0A&)"#+(DC"*'\$(MNO()*'+,-1*E(/'C1(@11+(A,10()#()*'+,-1*(%1+1,(@1)?11+(,>1&"1,(#@'&)1*"'9(/)-+-03%1*9(."&19(>\$'+),('+0('+".'\$,9(.'..'\$"'+(&1\$\$,("+(&A\$)A*19('+0(-#*
/A.'+(%1+1()/1*'>23((

49

!"#\$%&'()*+,-*)./*01,23)4/3,5/3.*6,-*)./*0,37+,5/3.*,8.*+,)3+9:9.:4:;*.,:(,.8'(8),37+
;/:.;/:)8.1,37+,<3=4*)9:;/3>*,?\$,4:,./:@,=:7='8.9A*'0,4/34,BC&,@3.,4/*,97(:)D349:7
D:'*=8'*,:(,/*)*+940E,&':7>,@94/,4/*,@:)F,:(,&A*)01,23=G*:+1,37+,2=53)401,4/*
-*)./*0H5/3.*,*I;*)9D*74,.*3'*+,4/*,87+*).437+97>,4/34,BC&,@3.,4/*,4)37.(:)D97>
D34*)93',37+,4/*,97(:)D349:7,D:'*=8'*,:(,/*)*+940E
!"J\$%K38',L*)>1,M37*4,2*)4N6,L*)>,8.*+,4/*,7*@'0,+9.=:A*)*+,*7+:78='*3.*,*7N0D*1,!"#OP1
4:,=84,QRST,BC&,37+,<3=4*)9:;/3>*,K\$\$,BC&1,37+,4/*7,8.*+,4*)D973',4)37.(*)3.*
*7N0D*,37+,BC&,'9>3.*,4:,)*U:97,4/*.*,.*;3)34*,;9*=*.,974:,:7*,;9*=*,:(,BC&E
5)*349:7,:(,4/*,(9).4,)*=:D<97374,BC&,D:'*=8'*,@3.,4/*,<*>97797>,:(,4/*,3>*,:(
<9:4*=/7:':>0E,?/*,7*@,D:'*=8'*,@3.,7:4,;'3=*+,97.9+*,3,D3DD3'937,=*'',<*=38.*,:(
=:7=*)7.,97,4/*,.=9*749(9=,=:DD87940,)*>3)+97>,>*7*49=,4)37.(*).E
!"JV%-*)<*)4,L:0*)1,Q437'*0,5:/*71,&779*,5/37>6,L*)>1,L:0*)1,37+,5:/*7,8.*+
!"#OP,4:,9.:'34*,37,9743=4,>*7*,(:),F373D0=97,)*.9.437=*E,L:0*)1,5:/*71,37+
5/37>,.;'9=*+,4/*,F373D0=97,)*.9.437=*,>*7*,974:,37,!"#OP,=84,;'3.D9+,4/34
3')*3+0,=:74397*+,4*4)3=0='97*,)*.9.437=*,37+,;):+8=*+,3,)*=:D<97374,<3=4*)93'
;'3.D9+,D:'*=8'*,@94/,+83',3749<9:49=,)*.9.437=*E,?/*0,4/*7,4)37.(:)D*+ !\$%"#&'
@94/,4/9.,*7>97**)*+,;'3.D9+E
!"JJ%W*7*74*=/1,P7=E6,?/*,(9).4,;):+8=4,:(,>*7*49=,*7>97**)97>1,4/*,>*7*,(:),/8D37,
.:D34:.43497,X/8D37,>):@4/,/:)D:7*Y)*'*3.97>,97/9<94:)0,(3=4:)Z1,@3.,*I;)*..*+,97,
<3=4*)93,37+,377:87=*+,<0,W*7*74*=/E
!"[T%ME,\E,W:)+:71,])37F,O8++'*6,W:)+:7,37+,O8++'*,.8==*..(8''0,D9=):97U*=4*+,7:)D3'
>*7*.,974:,D:8.*,>*)DY'97*,=*''.E
!"[\$%O9=/3)+,K3'D94*)1,O3';/,L)97.4*)6,K3'D94*),37+,L)97.4*),D9=):97U*=4*+,4/*,>*7*,(:)
)34,>):@4/,/:)D:7*,974:,D9=*,*D<)0:.E,?/9.,@3.,4/*,(9).4,>*7*49=,>*)DY'97*,^=8)*_,)*;:)4*+
97,3,D3DD3'E,?/*,)*=9;9*74,D:8.*,@3.,=3''*+,^'944'*_,<*=38.*,94,.8((*)*+,():D,3,(:)D
:(,=:7>*7943',+@3)(9.DE
!"[[%Q4*A*7,O:.*7<*)>6,O:.*7<*)>,37+,/9.,=:''*3>8*.,@*)*,>9A*7,3;;):A3',4:,;*)(:)D
4/*,(9).4,>*7*,4)37.(*),*I;*)9D*74,97,/8D37,;349*74.,.8((*)97>,():D,D*43.4349=
D*'37:D3E,?/9.,*I;*)9D*74,)*;)*.*74*+,>*7*49=,4)3=F97>,@94/,4/*,D3)F*),>*7*,C*:O
37+,7:4,>*7*,4/*)3;0E,
!""T%\E,])*7=/,&7+*).:71,29=/3*',L'3*.*1,`*77*4/,58'A*)6,&4,!\$6#\$,;EDE,:7,])9+301
Q*;4*D<*),!S1,!""T,34,4/*,C349:73',537=*),P7.49484*1,3,(:8),0*3),:'+,>9)'1,&./374/9,B*
Q9'A3,():D,5'*A*'37+1,a/9:1,<*=3D*,4/*,(9).4,/8D37,>*7*Y4/*)3;0,;349*74E,Q/*,@3.
97(8.*+,@94/,/*),:@7,@/94*,<'::+,=*''.,=3))097>,4/*,=:))*=4*+,A*).9:7,:(,4/*,3+*7:.97*
+*3D973.*,X&B&Z,>*7*E,B).E,&7+*).:71,L'3*.*,37+,58'A*),+9+,7:4,*I;*=4,D*3797>(8'
)*.8'4.,():D,4/*,*I;*)9D*74,(:),3<:84,!,0*3)E,&,.*=:7+,>9)'1,5074/93,584./3''1,@3.,
.9D9'3)'0,97U*=4*+,97,!""TE,O*;:)4.,97,M87*,!""V,./:@*+,4/*,4@:,>9)'.,@94/,.D9'*.,37+
=/9'+9./,*7*)>01,;'3097>,97,3,.=/::',03)+E,L:4/,>9)'.b,9DD87*,.0.4*D.,@*)*,@:)F97>
*((*=49A*'0E
!""S%a4/*),>*7*,4/*)3;0,=37+9+34*.,97='8+*,.9=F'*,=*'',37*D931,/*D:;/9'931,+93<*4*.1,
=37=*)1,37+,/*3)4,+9.*3.*,;349*74.E,W*)D,'97*,>*7*,4/*)3;0,9.,+*<34*+,+8)97>,D**497>,:(
4/*,O*=:D<97374,BC&,&+A9.:)0,5:DD944**E,L0,!""c,3,>):@97>,78D<*),:(,;):;:.3'.
3@394,)*A9*@,<0,4/*,-8D37,W*7*,?/*)3;0,Q8<=:DD944**,:(,4/*,O*=:D<97374,BC&
&+A9.:)0,5:DD944**E
!""#%G*+,<0,ME,5)39>,R*74*)1,3,>):8;,34,4/*,97.49484*,(:),W*7:D9=,O*.*3)=/,X?PWOZ,97
23)0'37+1,;8<'9./*+,4/*,(8'',>*7*,.*d8*7=*,:(,4/*,<3=4*)98D,()*#+,'&-.%'/0&-)/12)3 3
'37+D3)F,97,D9=):<9:':>9=3',)*.*3)=/,3.,4/*,(9).4,()**Y'9A97>,:)>379.D,@/:.*,>*7*49=
^<'8*;)974_,@3.,+*=:+*+E,,
!""c%&,D8'497349:73',=:''3<:)349:7,97='8+97>,D:)*,4/37,!TT,'3<:)34:)9*.,():D,e8):;*1

50

!"#\$%&'(')'\$%'()%*'+'(%,'-%./0%123-.%.4%5(3'607%./0%0(.230%80(490%-0:50(;0%41%'
05<'3=4.0\$%./0%=0'-.%!"##\$"%&'(#)*+#)%),-*-")>%!.+#)%),-*-") 2-%'%;49903;2'77=%-28(212?
;'(.%=0'-.%;4994(7=%5-0)%2(%@'<2(8%'()%2(%10390(.'.24(%41%'7;4/472;%@0603'80-%'()%2,2)07=%5-0)%2(%./0%7'@43'.43=%'-%'%94)07%438'(2-9%143%5()03-.'()2(8%;07757'3%'()
9470;57'3%+34;0--0-%41%05<'3=4.0->
ABBCD";20(.2-.-%70)%@=%E'(%F2795.%'.%";4.7'()G-%H4-72(%E(-.2.5.0%30+43.0)%./0%-5;;0--157
;74(2(8%41%'%-/00+\$%('90)%I477=\$%1349%./0%;077%41%'(%')57.%5))03%;077>%J/0%;74(2(8%41
I477=%-+'3<0)%2(.03('.24('7%)0@'.0%'@45.%0./2;'7%'()%943'7%2--50-%;4(;03(2(8%;74(2(8>
"5@-0:50(.7=\$%-;20(.2-.-%'.%";4.7'()G-%H4-72(%E(-.2.5.0\$%2(%;477'@43'.24(%,2./%";4.7'()?
@'-0)%KKL%J/03'+05.2;-\$%-5;;0--1577=%;74(0)%.,4%80(0.2;'77=%94)2120)%7'9@-\$%('90)
K477=%'()%M477=\$%./'.%,030%80(0.2;'77=%94)2120)%,2./%'%/59'(%80(0%-4%./'.%./023%927<
;4(.'2(0)%'%+34.02(%;'770)%1';.43%EN\$%'%@744)?;74..2(8%+34.02(%./'.%;'(%@0%0O.3';.0)%'()
5-0)%2(%.30'.2(8%/59'(%/094+/272'>
ABBPDQ603%BBR%41%./0%80(490%-0:50(;0%41%./0%123-.%957.2;07757'3%438'(2-9\$%./0%.2(=
345(),439%/")0&%\$"12-3-*+)4)5"0*\$%,'-%30+43.0)>%#7./458/%/.+)4)5"0* 2-%'%+3292.260
438'(2-9\$%2.%-/'30-%9'(=%41%2.-%0--0(.2'7%80(0.2;%'()%@247482;'7%;/'3';.032-.2;-%,2./
/59'(-%'()%9'=%/07+%-;20(.2-.-%2)0(.21=%'()%;/'3';.032S0%./0%80(0-%2(64760)%2(%/59'(
@24748=%'()%)2-0'-0>%%
";20(.2-.-%+34)5;0)%'%)0.'270)%'()%';;53'.0%+/=-2;'7%9'+\$%43%74;'.24(\$%143%94-.%41%./0
TU\$UUU%<(4,(%/59'(%80(0-\$%'%9270-.4(0%143%./0%V59'(%W0(490%K34X0;.>%%
YUUUD#%.0'9%70)%@=%E(84%K4.3=<5-%41%./0%",2--%Z0)03'7%E(-.2.5.0%41%J0;/(4748=%2(%[532;/
'()%K0.03%\0=03%41%./0%!(2603-2.=%41%Z302@538%2(%W039'(=%30+43.0)%./0%;30'.24(%41
80(0.2;'77=%94)2120)%32;0%;'770)%]847)0(%32;0^\$%,/2;/%;'(%+34)5;0%7'380%'945(.-%41
@0.'?;'34.0(0\$%'%-5@-.'(;0%./'.%/59'(%@02(8-%;'(%.53(%2(.4%_2.'92(%#>%]W47)0(%32;0^
;457)%'77062'.0%@72()(0--%;'5-0)%@=%62.'92(%#%)012;20(;=%2(%927724(-%41%+4603.=?-.32;<?
0(%+04+70%'345()%./0%,437)>
J/0%80(490%-0:50(;0%41%./0%1352.%17=%6%&*&7\$-4"+')4"0&5"*3)% ,'-%+5@72-/0)%./3458/
'%;477'@43'.24(%@0.,00(%'%+326'.0%;49+'(=\$%&0703'%W0(492;-\$%'()%30-0'3;/03-%,437)?
,2)0%-.5)=2(8%./0%1352.%17=>%%6.+')4"0&5"*3)%\$%'%94)07%5-0)%,2)07=%5-0)%2(%./0%7'@43'.43=\$
2-%./0%7'380-.%'(29'7%-4%1'3%.4%/'60%2.-%80(0.2;%;4)0%)0;2+/030)>%%
#%3458/%)3'1.%41%./0%/59'(%80(490%,'-%;49+70.0)%@=%'%.0'9%41%A`%2(.03('.24('7%
2(-.2.5.24(-%./'.%1439%./0%V59'(%W0(490%"0:50(;2(8%&4(-43.259>%H0-0'3;/03-%'.
&0703'%W0(492;-%'7-4%'((45(;0)%;49+70.24(%41%./023%a123-.%'--09@7=G%41%./0%80(490>%%

51

!""#\$%&'()*+,-+.'#!/'!""#/'0)1)+-'2)&34567'-&8'9:)';&9)+&-953&-1'<,4-&'2)&34)
=)>,)&65&?'03&73+95,4'@35&91.'-&&3,&6)8'9:)'A,*157:5&?'3B'9:)'&)-+1.'634A1)9)
7)>,)&6)'3B'9:)':,4-&'?)&34)'C'9:)'?)&)956'D*1,)A+5&9E'B3+'-':,4-&'*)5&?F'G:57
-6634A157:4)&9'933H'9:)'5&9)+&-953&-1'9)-4'-14379'9I)&9.'.)-+7'-&8'5&J31J)8'9:)'
6311-*3+-953&'3B'9:3,7-&87'3B'765)&95797'B+34'-+3,&8'9:)'I3+18F'0)1)+-'2)&34567
+)A3+9)8'634A1)95&?'9:)'I3+H'5&'-AA+3K54-9)1.'&5&)'43&9:7F'G:)'9I3'?+3,A7'85BB)+)8
5&'9:)5+')7954-9)7'B3+'9:)'&,4*)+'3B'?)&)7'5&'9:)':,4-&'?)&34)/'*,9'9:)'+-&?)'A+)8569C
)8'*.'*39:'?+3,A7/'*)9I))&'!L/"""'-&8'M"/"""'?)&)7/'57'B-+'B)I)+'9:-&'9:)'A+)J53,7
)7954-9)'3B'#""/"""'?)&)7F'G:57',&)KA)69)8'B5&85&?'7,??)79)8'9:-9'-&'3+?-&574'-7
634A1)K'-7'-':,4-&'*)5&?'6-&'*)'4-8)'3B'73'B)I'?)&)7/'3&1.'9I56)'-7'4-&.'-7'B3,&8
5&'9:)'I3+4'!F'"#"\$%&' 3+'9:)'B1.'()*+"#%&,\$%'-".F'G:)',&J)515&?'3B'9:)'B,11'7)>,)&6)
3B'9:)':,4-&'?)&34)'4-H)7'59'A3775*1)'B3+'+)7)-+6:)+7'-11'3J)+'9:)'I3+18'93'*)?5&
8)J)13A5&?'9+)-94)&97'B3+'4-&.'857)-7)7F'
N+)758)&9'2)3+?)'O,7:'8)658)8'9:-9'3&1.')KA)+54)&97'5&J31J5&?'9:)')K5795&?'PM')4*+.C
3&56'79)4'6)11'15&)7'I3,18'*)')15?5*1)'B3+'A3775*1)'B)8)+-1'B,&85&?F'G:)'A+)758)&9Q7
8)65753&'I-7'857-AA35&95&?'93'4-&.'765)&95797'I:3':3A)8'93',7)')4*+.3&56'79)4'6)117
93'8)J)13A'9+)-94)&97'B3+'4-&.'-514)&97F
R8J-&6)8'0)11'G)6:&313?./'-'74-11'634A-&.'5&'S-77-6:,7)997/'-&&3,&6)8'9:-9'59':-8
7,66)77B,11.'613&)8':,4-&')4*+.37'B3+'9:)'A,+A37)'3B')K9+-695&?'9:)5+'79)4'6)117F'G:57
4)9:38'63,18',1954-9)1.'*)',7)8'93'9+)-9'A-95)&97'I59:'-'J-+5)9.'3B'857)-7)7'*.'4-H5&?
+)A1-6)4)&9'6)117/'7,6:'-7'&)+J)'-&8'4,761)'6)117/'I:56:'6-&'*)'9+-&7A1-&9)8'*-6H'5&93
7-4)'A)+73&'I59:3,9'9:)'+57H'3B'*)5&?'+)@)69)8'*.'9:)'*38.F''
NNT'G:)+-A),9567/'9:)'634A-&.'9:-9':)1A)8'93'613&)'U311.'9:)'7:))A/'-&&3,&6)8'9:-9'59
:-8'613&)8'B5J)'?)&)956-11.'4385B5)8'A5?1)97'I59:'-&'5&-695J-9)8/'3+'DH&36H)8'3,9E/
?)&)'9:-9'I3,18'4-H)'9:)5+'3+?-&7'4,6:'1)77'15H)1.'93'*)'+)@)69)8'I:)&'9+-&7A1-&9)8
5&93'-':,4-&'+)65A5)&9F'G:)'7,66)77'3B'NNT'G:)+-A),9567'*+5&?7':3A)'93'9:)'9:3,7-&87
3B'A)3A1)'I:3'-+)'I-595&?'93'+)6)5J)'83&-9)8'3+?-&7'7,6:'-7':)-+97/'1,&?7/'H58&).7/'-&8
15J)+7F''
!""!\$U311.'9:)'7:))A/'9:)'B5+79'4-44-1'93'*)'613&)8'B+34'-&'-8,19'6)11/'8)J)13A)8'-+9:+5C
957'-9'-'+)1-95J)1.')-+1.'-?)'3B'B5J)'.)-+7F';9'57'&39'61)-+'I:)9:)+'U311.Q7'63&85953&'I-7
9:)'+)7,19'3B'-'?)&)956'8)B)69'6-,7)8'*.'613&5&?/'3+'I:)9:)+'59'I-7'-'4)+)'635&658)&6)F
G:)'&)I7':-7'+)&)I)8'8)*-9)7'3&'I:)9:)+'613&)8'-&54-17'-+)'7,76)A95*1)'93'A+)4-9,+)
-?5&?'-&8':)-19:'A+3*1)47'-&8':-7'-173'*))&'-'7)9*-6H'B3+'9:37)'I:3'-+?,)'9:-9'613&5&?
6-&'*)',7)8'93'?)&)+-9)'-'7,AA1.'3B'3+?-&7'93':)1A'A-95)&97'3&'9:)'9+-&7A1-&9'1579F''

52

## Appendix B Glossary of Terms

!"#\$

!"#\$%&'()*)(#+%&),*-"%.)-#)(/00#"-)1*2-'"%*+)3"#4-56
7#8-*%8()8/-"%'8-),%.-/"')#9)2*"1#5:&"*-'(;)*,%8#
*2%&(;)8/2+'#-%&'(;)(*+-(;)*8&)\$%-*,%8(6

!%&'(')&'*+,-.-*&')%

<(')#9)*8)*8-%1%#-%2)-#)('+'2-)1*2-'"%*)2#8-*%8%83)-5')=>?
#9)%8-'"'(-6)@5')0ABC)0+*(,%&)=>?)2#8-*%8()-5')3'8')9#"
1'-*D+*2-*,*(')-5*-)0"#\$%&'()"'(%(-*82')-#)-5')*8-%1%#-%2
*,0%2%++%86)C82')1*2-'"%*)*"')-"*8(9#",'&)4%-5)-5')0ABC
0+*(,%&;)-5':)1'3%8)0"#&/2%83)*8&)('2"'-%83)1'-*D+*2-*,*('
0"#-'%86)E'2"'-'&)1'-*D+*2-*,*(')1"'*F()&#48)*,0%2%++%8;
"'8&'"%83)-5')*8-%1%#-%2)5*",+'(()-#)-5')1*2-'"%*+)5#(-6)C8+:
1*2-'"%*)2#8-*%8%83)-5')0ABC)0+*(,%&)2*8)3"#4)*8&)9#",
2#+#8%'()%8)8/-"%'8-),'&%/,)2#8-*%8%83)*,0%2%++%8;)45%+'
/8-"*8(9#",'&)2'++()-5*-)5*\$')8#-)-*F'8)/0)-5')0ABC)
0+*(,%&)2*88#-)3"#4)#8)-5')*,0%2%++%8)('+'2-%#8)0+*-'(6

!\$#('%)/-

?)2*"1#5:&"*-')%(#+*-'&)9"#,)0+*8-()-5*-)%()8#",*++:
/('&)*()(#/"2')#9)9##&)1:)1*2-'"%*6

0-&#12#*&#3#/-

G'-*D+*2-*,*(')%()*)0"#-'%8)-5*-)0"#\$%&'()"'(%(-*82')-#
-5')*8-%1%#-%2)*,0%2%++%86)@5')1'-*D+*2-*,*(')0"#-'%8)%()
0"#&/2'&)*8&)('2"'-'&)1:)1*2-'"%*)-5*-)5*\$')1''8)
-"*8(9#",'& 4%-5)*)0+*(,%&)2#8-*%8%83)-5')3'8')9#")
1'-*D+*2-*,*('6)@5')('2"'-'&)1'-*D+*2-*,*(')%8*2-%\$*-'(
-5')*,0%2%++%8)0"'('8-)%8)-5')BG)8/-"%'8-)*3*";)45%25
*++#4()9#")1*2-'"%*+)3"#4-5)*8&)'.0"'((%#8)#9)8'4+:
*2H/%"'&)3'8'()*+(#)2#8-*%8'&)#8)-5')0+*(,%&;)!"#"\$ AI!6

0')&-*4%).)"5

?00+:%83)1%#+#3:)%8)-5')"'*+)4#"+&)1:)-5')(0'2%9%2
,*8%0/+*-%#8)#9)+%\$%83)#"3*8%(,(;)'(0'2%*++:)*-)-5'
3'8'-%2)+'\$'+;)-#)0"#&/2')0#-'8-%*++:)1'8'9%2%*+)0"#&/2-(6

6.)%'%"

7+#8%83)%()-5')0"#2'(()#9)3'8'"*-%83)\$%"-/*++:)'8&+'((
2#0%'()#")2+#8'()#9)*8)#"3*8%(,)#")('3,'8-)#9)=>?6
7+#8%83)0"#&/2'()*)0#0/+*-%#8)-5*-)5*()*8)%&'8-%2*+
3'8'-%2),*F'/06

6).)%5

?)2+/,0)#9)3'8'-%2*++:)%&'8-%2*+)1*2-'"%*+)2'++()3"#4%83
#8)*8)*3*")0+*-'6)G'2*/(')*++)-5')2'++()%8)*)(%83+'
2#+#8:)*"')3'8'-%2*++:)%&'8-%2*+;)-5':)*"')2*++'&)2+#8'(6

67.&7\$-+8-9'#

@5')+%H/%&)*8&)(#+%&),'&%*)"'9'""'&)-#)*()BG)J8*,'&
*9-'")B/"%*)*8&)G'"-*8%K)1"#-5)*8&)*3*")*"'),*&')9"#,
*8)'.-"*2-)#9):'*(-)*8&)*8)'8L:,*-%2)&%3'(-)#9),'*1:0"#&/2-( 45%25)0"#\$%&')*),%.-/"')#9)2*"1#5:&"*-'(;
*,%8#)*2%&(;)8/2+'#-%&'(;)(*+-(;)*8&)\$%-*,%8(;)*++)#9
45%25)*"')8/-"%'8-()9#")1*2-'"%*+)3"#4-56)?3*";)45%25)%(
9"#,)('*4''&;)0#+:,'"%L'()45'8)5'*-'&)*8&)2##+'&)-#
9#",)*)(#+%&)3'+)J(%,%+*")-#)M'++DC)3'+*-%8K;)*8&)
9/82-%#8()-#)0"#\$%&')*)(#+%&)(/00#"-)#8)45%25)-#)2/+-/"'
-5')1*2-'"%*6

53

!"#"\$%&'(#)%#""*%#)

!"#\$%&'()*+&,(-'\$-.\$&'\$-/0&'(1%21\$0#'#,(3\$%&,#/(&+
45678\$9:\$(',/-;*3('0\$-/\$#+(%('&,('0\$1)#3(.(3\$0#'#1<

!"#"'+"),-.\$%/#

=#'#\$#>)/#11(-'\$('\$&++\$-/0&'(1%1\$(1\$3&/#.*++:\$/#0*+&,#;
,-\$&++-?\$.-/\$;(..#/('0\$3-';(,(-'1\$&';\$,-\$)/#@#',\$?&1,#.*+
-@#/)/-;*3,(-'\$-.\$*''##;#;\$)/-,#('1<\$!"#\$0#'#1
('@-+@#;\$('\$,"#\$,/&'1)-/,\$&';\$9/#&A;-?'\$-.\$.--;\$&/#
0--;\$#>&%)+#1\$-.\$"(0"+:\$/#0*+&,#;\$0#'#1<\$B-/\$#>&%)+#C
,"#\$1(%)+#\$1*0&/C\$&/&9('-1#C\$3&'\$9#\$*1#;\$&1\$&\$1-*/3#\$-.
#'#/0:\$&';\$3&/9-'\$9:\$9&3,#/(&<\$!"#\$9&3,#/(&+\$#'D:%#1
,"&,\$&/#\$'##;#;\$,-\$9/#&A\$;-?'\$-/\$;(0#1,\$&/&9('-1#\$.-/
.--;\$&/#\$'-,\$#>)/#11#;\$('\$,"#\$&91#'3#\$-.\$&/&9('-1#\$9*,
&/#\$#>)/#11#;\$?"#'\$&/&9('-1#\$(1\$)/#1#',\$('\$,"#
#'@(/-'%#',< E'\$-,"#/\$?-/;1\$?"#'\$&/&9('-1#\$(1
&/-*';C\$,"#\$0#'#1\$.-/\$,"#1#\$;(0#1,(@#\$#'D:%#1\$&/#
,*/'#;\$-'<\$F"#'\$&/&9('-1#\$/*'1\$-*,\$,"#1#\$0#'#1\$&/#
,*/'#;\$9&3A\$-..<\$G##\$7))#';(>\$5\$.-/\$&\$%-/#\$;#,&(+#;
#>)+&'&,(-'\$-.\$,"#\$/-+#\$,"&,\$&/&9('-1#\$)+&:1\$('\$,"#
/#0*+&,(-'\$&';\$#>)/#11(-'\$-.\$,"#\$=/##'\$B+*-/#13#',
H/-,#('\$0#'#<

!*""#'0-,/*"1&"#\$'2*/\$"%#

=/##'\$B+*-/#13#',\$H/-,#('\$4=BH8\$?&1\$-/(0('&++:\$(1-+&,#;
./-%\$,"#\$9(-+*%('#13#',\$I#++:.(1"C\$!"#\$%&"'()*+,%&*'<
!"#\$0#'#\$.-/\$=BH\$"&1\$/#3#',+:\$9##'\$3+-'#;<\$!"#
*'(J*#\$,"/##K;(%#'1(-'&+\$3-'.-/%&,(-'\$-.\$=BH\$3&*1#1
(,\$,-\$/#1-'&,#\$?"#'\$#>)-1#;\$,-\$*+,/&@(-+#,\$+(0",\$&';\$0(@#
-..\$#'#/0: ('\$,"#\$.-/%\$-.\$@(1(9+#\$0/##'\$+(0",<\$\$

2-.13%4

7\$3(/3*+&/\$567\$%-+#3*+#C\$3&)&9+#\$-.\$1#+.K/#)+(3&,('0C
3&//:('0\$-'#\$-/\$%-/#\$0#'#1\$.-/\$&',(9(-,(3\$/#1(1,&'3#
)/-,#('1\$&';\$&\$3+-'#;\$.-/#(0'\$0#'#\$1*3"\$&1\$=BH<

5!67

H+&1%(;\$3-',&('('0\$,"#\$=BH\$1#J*#'3#\$&';\$&%)(3(++('
/#1(1,&'3#\$0#'#C\$?"(3"\$3-;#1\$.-/\$9#,&K+&3,&%&1#<

+"&/38%#.#\$'9:;'<"&=#/-/)>

!"#\$)/-3#11\$-.\$3*,,('0\$&';\$/#3-%9('('0\$567\$./&0%#',1
&1\$&\$%#&'1\$,-\$(1-+&,#\$0#'#1\$-/\$,-\$&+,#/\$,"#(/\$1,/*3,*/#
&';\$.*'3,(-'<

?&*""#%#)

H/-3#11\$-.\$(;#',(.:('0\$?&',#;\$9&3,#/(&\$./-%\$&\$9&3,#/(&+
+(9/&/:<

?\$"*%-"'<"&=#%@,"

L('(%(D('0\$,"#\$)-11(9(+(,:\$-.\$-*,1(;#\$9&3,#/(&+\$
3-',&%('&,(-'\$;*/('0\$&'\$#>)#/(%#',\$,"/-*0"\$
-91#/@&'3#\$-.\$3+#&'+('#11\$&';\$*1('0\$3&/#.*+\$+&9-/&,-/:
,#3"'(J*#1<

?\$*".A%#)

H/-3#11\$-.\$)&11('0\$&'\$('-3*+&,('0\$+--)\$?(,"\$9&3,#/(&
-'\$(,\$&3/-11\$&'\$&0&/\$)+&,#

B"&\$/*

7'\$&*,-'-%-*1+:\$/#)+(3&,('0\$567\$%-+#3*+#C\$1*3"\$&1
&\$)+&1%(;C\$(',-\$?"(3"\$.-/#(0'\$567\$./&0%#',1\$&/#\$
('1#/,#;\$&';\$,"#'\$)/-)&0&,#;\$('\$&\$"-1,\$3#++<

54

## Appendix C Basic Molecular Biology Concepts and

Terminology
!"#\$%&'"()"\$*+",-.-/0"1(2,&"2+.+3,#"\$*3\$"3,,",-.-/0"(203/-#4#"(203/-5+"\$*+4#+,.+#"-/
#(4+"%/-6%+")3#*-(/7"!"&+\$3-,+&"8,%+92-/\$"()"\$*-#"(203/-53\$-(/"-#"93##+&"(/"\$("())#92-/07
:+,,#"32+"\$*+"#43,,+#\$")%/;\$-(/3,"%/-\$#";3938,+"()"-/&+9+/&+/\$"2+92(&%;\$-(/7"<3/'
83;\$+2-3=")(2"-/#\$3/;+=";3/"#%2.-.+"3#"#-/0,+";+,,#7">*+";*+4-;3,"4(,+;%,+#"1-\$*-/"+3;*";+,,
32+"(203/-5+&"\$("9+2)(24"-/";(/;+2\$7
!"##\$%&'(%)"%*+,-(%.(%&/#0/+"%'(1%2'+3"\$0"1
:+,,#";3/"8+"03\$*+2+&")2(4"\$*+-2"/3\$%23,",(;3\$-(/#"3/&"02(1/"-/#-&+",38(23\$(2'";(/\$3-/+2#7
!992(92-3\$+")((&"3/&"+/.-2(/4+/\$"4%#\$"8+"92(.-&+&")(2"\$*+";+,,#"\$("02(17"?3;\$+2-3"3/&
'+3#\$"32+".+2'"+3#'"\$("02(1"-/";%,\$%2+7":+,,#"\$3@+/")2(4"9,3/\$#="-/#+;\$#"3/&"3/-43,#";3/"3,#(
8+"02(1/="8%\$"32+"4(2+"&-))-;%,\$"\$(";32+")(27
!)\$+2"02(1\$*"-#";(49,+\$+=";+,,#"-/";%,\$%2+";3/"8+"*32.+#\$+&"3/&"#\$%&-+&7
!#,(.(*
A*+/"3"9(9%,3\$-(/"()";+,,#"-#"92+932+&"8'"02(1\$*")2(4"3"#-/0,+";+,,="3,,"\$*+";+,,#"-/"\$*+"
9(9%,3\$-(/"1-,,"8+"0+/+\$-;3,,'"-&+/\$-;3,7"B%;*"3"9(9%,3\$-(/"-#";3,,+&";,(/3,7">*+"92(;+##"()
;2+3\$-/0"3";,(/3,"9(9%,3\$-(/"-#";3,,+&";,(/-/07">*+"9%29(#+"()"#\$2+3@-/0"83;\$+2-3"(/"3032"-#
\$("0+/+23\$+"#-/0,+";(,(/-+#="+3;*"32-#-/0")2(4"3"#-/0,+";+,,7
4,,5.(*%.(\$.1"%&"##\$
>*+"4(,+;%,+#"-/#-&+"3";+,,"+3;*"9+2)(24"3"0-.+/")%/;\$-(/7"C(2"-/#\$3/;+="DE!
4(,+;%,+#"#\$(2+"-/)(243\$-(/"F,-@+"\$*+"*32&"&2-.+"-/"3";(49%\$+2G7"H2(\$+-/#"32+"\$*+"1(2@*(2#+#
()"\$*+";+,,7
>("#\$%&'"\$*+#+"4(,+;%,+#"1+"92+932+"3";,(/3,"9(9%,3\$-(/")2(4"3";+,,"\$'9+"()"-/\$+2+#\$=
82+3@"(9+/"\$*+";+,,#"3/&"#(2\$"\$*+";(/\$+/\$#7"C(2"-/#\$3/;+="-\$"-#")3-2,'"+3#'"\$("#+9323\$+"3,,"\$*+"
92(\$+-/#")2(4"3,,"\$*+"DE!"4(,+;%,+#7
H%2-)'-/0"3"#-/0,+"#9+;-+#"()"92(\$+-/"(%\$"()"\$*+"4-I\$%2+"()"92(\$+-/#")(%/&"-/#-&+"3";+,,
\$'9+"-#"3,#("9(##-8,+7"J3;*"\$'9+"()"92(\$+-/"*3#"%/-6%+"9*'#-;3,"3/&";*+4-;3,"92(9+2\$-+#7
>*+#+"92(9+2\$-+#"3,,(1"\$*+"#+9323\$-(/"()"92(\$+-/"#9+;-+#"83#+&"(/"#-5+=";*320+="(2
*'&2(9*(8-;-\$'=")(2"-/#\$3/;+7
67"&.'#%8,#"&/#"\$9%\$7"&.'#.:"1%;/(&0.,(\$
A+"1-,,"\$3@+"3";,(#+",((@"3\$"\$*2++".+2'"#9+;-3,"@-/&#"()"4(,+;%,+#")(%/&"-/#-&+";+,,#K
DE!="LE!"3/&"92(\$+-/#7"J3;*"()"\$*+#+"4(,+;%,+#"9+2)(24#"3"&-))+2+/\$")%/;\$-(/7"DE!
4(,+;%,+#"32+",-@+")-,+";38-/+\$#"-/"1*-;*"-/)(243\$-(/"-#"#\$(2+&7"LE!"*+,9#"\$("2+\$2-+.+"3/&
+I+;%\$+"\$*+"-/#\$2%;\$-(/#"1*-;*"32+"#\$(2+&"-/"DE!7"H2(\$+-/#"32+"&+#-0/+&"\$("9+2)(24
;*+4-;3,";*(2+#"-/#-&+"F3/&"()\$+/"(%\$#-&+G"\$*+";+,,7
<=>?@2"%/(.3"+\$'#%0"87#'0"%;,+%).,#,*.&'#%.(;,+8'0.,(
>*+"43#\$+2"#;2-9\$")(2"+3;*"(203/-#4"-#"+/;(&+&"1-\$*-/"-\$#"&+(I'2-8(/%;,+-;"3;-&
FDE!G7">*+"-/)(243\$-(/"1-\$*-/"\$*+"DE!"4(,+;%,+M#"()"+3;*";+,,"-#"#%))-;-+/\$"\$("-/-\$-3\$+
+.+2'")%/;\$-(/"\$*3\$";+,,"1-,,"9+2)(247
DE!"4(,+;%,+#"32+".+2'",(/0";*3-/#";(49(#+&"()"2+9+3\$-/0"#%8%/-\$#7"J3;*"#%8%/-\$
F/%;,+(\$-&+G";(/\$3-/#"(/+"()")(%2"9(##-8,+"83#+#"92(\$2%&-/0")2(4"-\$#"#-&+K
3&+/-/+"F>G"
\$*'4-/+"F@G

;'\$(#-/+"F!G
0%3/-/+"FAG

55

!"#\$%&#'\$(%)*"+%,&-.%&/)"#%+&0%-+1*)1*-"(2&-&()#3&,*.-#+&)4&567&%,,%#*"-((8&\$)#,",*,&)4&\$0%9"\$-(&:-\$;:)#%&<"*0&:-,%,&=.)*.'+"#3&-()#3&"*,&,"+%>&?0%&"#4).9-*")#&\$-.."%+&:8&*0",
9)(%\$'(%&",&%#\$)+%+&"#&*0%&,%@'%#\$%&)4&*0%&:-,%,&!2&"2&#2&-#+&\$ -()#3&"*,&(%#3*0>
%&'()*+,-.(,)/&01-2)-&)1&-()34&+-)56!)2-,+7-+,(
A>&& B%\$-',%&*0%&,':'#"*,&)4&567&\$0-"#,&-.%&/)"#%+&0%-+1*)1*-"(2&*0%&,%@'%#\$%&",&+".%\$*")#-(
"\$#!!>&B8&\$)#C%#*")#2&<%&<."*%&567&,%@'%#\$%&4.)9&*0%&4.%%&DE&%#+&)4&*0%&:-\$;:)#%
-#+&<).;&)'.&<-8&*)<-.+&*0%&)*0%.&4.%%&%#+&FGEH>
!"#" DE>>>!!#\$">>>GE
I> ?0%&=.)*.'+"#3&:-,%,&-()#3&*0%&\$0-"#&-.%&4.%%&*)&4).9&,=)#*-#%)',&08+.)3%#&:)#+,
<"*0&-C-"(-:(%&:-,%,&)#&)*0%.&567&,*.-#+,&-\$\$).+"#3&*)&*0%&4)(()<"#3&.'(%,J
F"H&&&&&&&! =-".,&<"*0&\$
F""H&&&&&&# =-".,&<"*0&"
B%\$-',%&)4&*0%,%&.'(%,2&! -#+&\$ -.%&,-"+&*)&:%&\$)9=(%9%#*-.8&:-,%,K&" -#+&# -.%&-(,)
\$)9=(%9%#*-.8>
F"""H&&&&&&L).&*<)&567&,*.-#+,&*)&=-".&'=2&*0%8&9',*&:%&\$)9=(%9%#*-.8&-#+&.'#&"#
)==),"*%&+".%\$*")#,>&
!"#" FDE>>>!""\$#>>>GEH&\$-#&=-".&<"*0&FDE>>>"!##\$>>>GEH>&?0%,%&*<)&,*.-#+,&0-C%&
\$)9=(%9%#*-.8&,%@'%#\$%,>&?0%&+)':(%1,*.-#+%+&=-".&",&<."**%#&-,&4)(()<,J
DE>>>!""\$#>>>GE
GE>>>\$##!">>>DE
?0%&-:)C%&9)(%\$'(%&\$)#*-"#,&4"C%&:-,%&=-".,>&M#+%%+2&"#&#-*'.%2&567&-(9),*&-(<-8,
)\$\$'.,&"#&+)':(%1,*.-#+%+&4).92&*0%&*<)&,*.-#+,&\$)#*-"#"#3&\$)9=(%9%#*-.8&,%@'%#\$%,>
G> 567&9)(%\$'(%,&-.%&*8="\$-((8&*0)',-#+,2&,)9%*"9%,&9"((")#,&)4&:-,%&=-".,&()#3>
!)9%*"9%,&*0%&*<)&%#+,&)4&-&567&9)(%\$'(%&-.%&/)"#%+&*)&4).9&\$".\$'(-.&567>
N> 5)':(%1,*.-#+%+&5672&"#&"*,&#-*"C%&4).92&)\$\$'.,&-,&-&\$)"(%+&,=."#32&).&0%("O>&B%\$-',%&"*
",&*<)1,*.-#+%+2&"*&",&)4*%#&.%4%..%+&*)&-,&-&+)':(%&0%("O>
?0%&-.\$0"*%\$*'.%&)4&567&-(()<,&4).&-&C%.8&,"9=(%&,*.-*%38&+'."#3&.%=.)+'\$*")#J&?0%
*<)&,*.-#+,&)4&%-\$0&567&9)(%\$'(%&'#<"#+&-#+&P'#Q"=PK&*0%#2&%-\$0&,*.-#+&-(()<,&-&#%<
\$)9=(%9%#*-.8&\$)=8&)4&"*,%(4&*)&:%&9-+%&:8&-#&%#Q89%&\$-((%+&567&=)(89%.-,%>&?0",
.%,'(*,&"#&*<)&+-'30*%.&9)(%\$'(%,2&%-\$0&+)':(%1,*.-#+%+2&-#+&%-\$0&"+%#*"\$-(&*)&*0%&=-.%#*
9)(%\$'(%>
8,&-(012)319):6!)3,()-.();&,<.&,2(2)&*)-.()7(==
?0%&:")\$0%9",*.8&)4&("4%&.%@'".%,&0'#+.%+,&)4&C%.8&,=%\$"4"\$&-#+&%44"\$"%#*&\$0%9"\$-(
"#*%.-\$*")#,2&-((&0-==%#"#3&,"9'(*-#%)',(8>&?0%&9-/).&=(-8%.,&"#&*0%,%&"#*%.-\$*")#,&-.%
,0).*1("C%+&=.)*%"#&-#+&R67&9)(%\$'(%,&<0"\$0&\$-#&<).;&*)3%*0%.&).&"#+%=%#+%#*(8&*)
,%.C%&-&C-."%*8&)4&4'#\$*")#,>&S";%&5672&R67&-#+&=.)*%"#,&-.%&-(,)&()#3&\$0-"#,&)4
.%=%-*"#3&'#"*,>
:6!
R67&F.":)#'\$(%"\$&-\$"+H2&(";%&5672&\$)#,",*,&)4&4)'.&*8=%,&)4&:'"(+"#3&:()\$;,&,*.'#3
*)3%*0%.&"#&-&\$0-"#>&M*&+"44%.,&4.)9&567&"#&*0%&4)(()<"#3&.%,=%\$*,J

56

!"#\$%&'(\$)*+#+\$,-\$./0\$*(#\$!1\$"1\$#1\$*-2\$\$ 3'(*4,567\$8"#\$9*,(,-:\$('5#+\$*(#\$8"#\$+*;#\$*+\$%&(
</0\$#=4#98\$8"*8\$! 9*,(+\$>,8"\$\$?\$058"&':"\$./0\$4*-\$9*,(\$>,8"\$4&;95#;#-8*(@\$./0\$&(
</01\$,-\$4#55+\$./0\$,+\$'+'*55@\$+,-:5#A+8(*-2#2?\$!"#\$+':*(\$,-\$8"#\$./0\$)*4B)&-#\$,+\$(,)&+#1
-&8\$2#&=@(,)&+#?\$./0\$;&5#4'5#+\$*(#\$:#-#(*55@\$+"&(81\$4&;9*(#2\$8&\$</0\$;&5#4'5#+7\$8",+\$,+
)#4*'+#\$#*4"\$./0\$,+\$,8+#5%\$*\$4&9@\$&%\$*\$+"&(8\$+#:;#-8\$%(&;\$*\$</0\$;&5#4'5#?\$!"#\$9(&4#++
&%\$4&9@,-:\$+#:;#-8+\$&%\$</0\$,-8&\$./0\$,+\$4*55#2\$8(*-+4(,98,&-1\$*-2\$,+\$9#(%&(;#2\$)@\$*\$
9(&8#,-\$4*55#2\$./0\$9&5@;#(*+#?
%&'()*+,
C(&8#,-+\$3;&(#\$9(#4,+#5@1\$9&5@9#98,2#+6\$*(#\$*5+&\$5&-:1\$4"*,-A5,B#\$;&5#4'5#+\$)'8\$*(#\$;&(#
+8('48'(*55@\$2,D#(+#\$8"*-\$#,8"#(\$</0\$&(\$./0?\$!",+\$,+\$)#4*'+#\$8"#\$+')'-,8+\$&%\$9(&8#,-+1\$4*55#2
*;,-&\$*4,2+1\$4&;#\$,-\$8>#-8@\$2,%%#(#-8\$8@9#+?\$!"#\$#=*48\$+#E'#-4#\$&%\$*;,-&\$*4,2+\$*5&-:\$*
9&5@9#98,2#\$4"*,-\$2#8#(;,-#+\$"&>\$8"*8\$4"*,-\$>,55\$%&52\$,-8&\$,8+\$8"(##A2,;#-+,&-*5\$+8('48'(#?
!"#\$9(#4,+#\$8"(##A2,;#-+,&-*5\$%#*8'(#+\$&%\$8",+\$+8('48'(#1\$,-\$8'(-1\$2#8#(;,-#\$,8+\$%'-48,&-?
F"*8\$*\$9(&8#,-\$>,55\$-' 2#9#-2+\$&-\$8"#\$#=*48\$,)./)+0) &%\$,8+\$*;,-&\$*4,2+?
G-\$;&+8\$4*+#+1\$*\$9(&8#,-\$>,55\$9#(%&(;\$*\$+,-:5#\$%'-48,&-?\$H#(@\$2,D#(+#\$%'-48,&-+\$4*)#\$9#(%&(;#2\$)@\$9(&8#,-+I\$J&;#\$9(&8#,-+1\$4*55#2\$#-K@;#+1\$*48\$*+\$4*8*5@+8+\$,-\$4"#;,4*5
(#*48,&-+7\$+&;#\$4*((@\$+,:-*5+\$%(&;\$&-#\$9*(8\$&%\$*\$4#55\$8&\$*-&8"#(L&(1\$,-\$8"#\$4*+#\$&%
M"&(;&-#+N1\$%(&;\$&-#\$4#55\$8&\$*-&8"#(7\$+&;#\$9(&8#,-+\$3*-8,)&2,#+6\$"*D#\$8"#\$8*+B\$&%
%,:"8,-:\$,-8('2#(+7\$;*-@\$)#4&;#\$,-8#:(*5\$9*(8+\$&%\$8"#\$D*(,&'+\$9"@+,4*5\$+8('48'(#+\$,-+,2#
4#55+7\$*-2\$+8,55\$&8"#(+\$3(#:'5*8&(@\$9(&8#,-+6\$9&5,4#\$D*(,&'+\$*48,D,8,#+\$>,8",-\$4#55+\$+&\$*+\$8&
B##9\$8"#;\$>,8",-\$M5#:*5N\$5,;,8+?
1*+)2&30'-)43(5&))6-*7)+,*'+2830'+,)./)+0),
</0\$,+\$8"#\$9(,;*(@\$2#9&8\$%&(\$,-%&(;*8,&-\$,-\$5,D,-:\$+@+8#;+?\$0+\$;#-8,&-#21\$8",+
,-%&(;*8,&-\$,+\$5,-#*(1\$!"#"1\$#-4&2#2\$,-\$8"#\$+#E'#-4#\$&%\$!1\$"1\$#1\$9 )',52,-:\$)5&4B+\$*5&-:
8"#\$</0\$;&5#4'5#?\$!",+\$5,-#*(\$4&2#\$4*-\$)#\$9*++#2\$&-\$8&\$&%%+9(,-:L)#4*'+#\$</0\$4*)#\$(#95,4*8#2\$,-\$#=*48\$4&9,#+?
J"&(8\$+#:;#-8+\$&%\$#*4"\$</0\$;&5#4'5#\$*(#\$4"&+#-\$%&(\$8(*-+4(,98,&-\$*8\$*-@\$:,D#-\$8,;#?
!"#+#\$+#:;#-8+\$*(#\$4*55#2\$:#-#+?\$!"#\$#-K@;#\$./0\$9&5@;#(*+#\$4&9,#+\$8"#\$#-8,(#\$+#:;#-81
)*+#\$)@\$)*+#1\$*++#;)5,-:\$*-\$./0\$;&5#4'5#\$>",4"\$4&-8*,-+\$*\$+#E'#-4#\$&%\$!1\$"1\$# *-2\$\$
#=*485@\$4&;95#;#-8*(@\$8&\$8"#\$</0\$+#E'#-4#\$&%\$8"#\$8(*-+4(,)#2\$:#-#?
G-\$*22,8,&-\$8&\$9(&D,2,-:\$*\$;*+8#(\$8#;95*8#\$%&(\$4&9@,-:\$./0+1\$</0\$*5+&\$4&-8*,-+
+#E'#-4#\$,-%&(;*8,&-\$>",4"\$8#55+\$8"#\$./0\$9&5@;#(*+#\$>"#(#\$8&\$+8*(8\$8(*-+4(,),-:\$*\$:#-#
39(&;&8#(6\$*-2\$>"#(#\$8&\$+8&97\$"&>\$;*-@\$4&9,#+\$,8\$+"&'52\$;*B#\$*-2\$>"#-7\$*-2\$,8\$4*#D#-\$#;)#2\$4#(8*,-\$,-%&(;*8,&-\$>,8",-\$8"#\$./0\$+#E'#-4#\$8&\$2#8#(;,-#\$8"#\$5&-:#D,8@\$*-2
9(&2'48,D,8@\$&%\$8"*8\$./0?
!"#(#\$*(#\$8"(##\$;*O&(\$45*++#+\$&%\$./0+\$4&9,#2\$&%%\$</0\$8#;95*8#+I\$;#++#-:#(\$./0+1
&(\$;./0+1\$>",4"\$(#5*@\$8"#\$+#E'#-4#\$,-%&(;*8,&-\$(#E',(#2\$%&(\$*++#;)5,-:\$9(&8#,-+7\$8(*-+A
%#(\$./0+1\$&(\$8./0+1\$>",4"\$>&(B\$,-\$8"#\$*++#;)5@\$5,-#\$%&(\$9(&8#,-+7\$*-2\$./0+\$>",4"\$9#(A
%&(;\$+8('48'(*5\$%'-48,&-+?\$P&(\$#=*;95#1\$(,)&+&;*5\$./0+1\$&(\$(./0+1\$"#59\$)',52\$8"#
+4*%%&52,-:\$%&(\$(,)&+&;#+1\$8"#\$%*48&(,#+\$>"#(#\$9(&8#,-+\$*(#\$*++#;)5#2?
;./0+\$4*((@\$8"#\$+#E'#-4#\$,-%&(;*8,&-\$%&(\$;*B,-:\$9(&8#,-+?\$.,)&+&;#+\$(#*2\$8",+
+#E'#-4#\$&%\$-'45#&8,2#+1\$)@\$*\$9(&4#++\$4*55#2\$M8(*-+5*8,&-N1\$,-8&\$*\$+#E'#-4#\$&%\$*;,-&\$*4,2+?
Q&>\$,+\$8",+\$*44&;95,+"#2R\$!"#(#\$*(#\$&-5@\$%&'(\$B,-2+\$&%\$-'45#&8,2#+1\$)'8\$8>#-8@\$B,-2+\$&%
*;,-&\$*4,2+?
<'(,-:\$8(*-+5*8,&-1\$8"#\$(,)&+&;#\$(#*2+\$S\$-'45#&8,2#+\$*8\$*\$8,;#\$*-2\$*++,:-+\$*-\$*;,-&
*4,2\$8&\$#*4"\$+'44#++,D#\$8(,95#8?\$:'(); !(,95#8+\$*(#\$&%8#-\$(#%#((#2\$8&\$*+\$0'-'+,?\$T*4"\$*;,-&

57

!"#\$%#&%'()*%!''!"()\$%'+%'()%)*\$%+,%'()%-.+/#*-%0.+')#*%"(!#*1%2().)%!.)%34%0+&&#56)%'.#06)'&7
+.%"+\$+*&1%2(8&7%'()%6#*)!.%#*,+.9!'#+*%.)&#\$#*-%#*%:;<%#&%8&)\$%'+%!&&)956)%!%6#*)!.
&)=8)*")%+,%!9#*+%!"#\$&%#*%!%0.+')#*1%2(#&%&)=8)*")7%#*%'8.*7%!"##\$%&'&()"*&\$'+&\$!,-\$'+,'
.(/'&"*\$!"##\$0/#%\$"*'/\$,\$.(&1"2&\$2+,.& /#'(%"(!.!"').#&'#"%"()9#"!6%0.+0).'#)&1
>*%&899!.?7%'()%0.#9!.?%'.!*&,).%+,%#*,+.9!'#+*%/#'(#*%")66&%,+66+/&%'()%+.\$).@
:;<A;<BAC2D>;2A<>2
<6'(+8-(%'()%#*,+.9!'#+*%#'&)6,%#&%6#*)!.7%'()%#906#"!'#+*&%!.)%'(.))E\$#9)*&#+*!61%<%
,8*\$!9)*'!6%!&&890'#+*%+,%.)"+95#*!*'%:;<%')"(*+6+-?%#&%'(!'%0).9!*)*'%!*\$%\$)&#.!56)
"(!*-)&%#*%'()%,8*"'#+*#*-%+,%6#F#*-%")66&%"!*%5)%!""+906#&()\$%5?%"(!*-#*-%'()%6#*)!.
&)=8)*")%+,%'()#.%:;<1
3&*&2\$,(&\$%"21(&'&\$0"#&2\$/0\$456\$"*0/(),'"/*
<%-)*)%#&%!%&)-9)*'%/#'(#*%!%:;<%9+6)"86)%&#*-6)\$%+8'%,+.%"+0?#*-%#*'+%A;<1
:#.)"'6?%+.%#*\$#.)"'6?7%'(#&%A;<%/#66%0).,+.9%!%,8*"'#+*1%>'%#&%"+*F)*#)*'%'+%'(#*G%+,%!%-)*)7
'().),+.)7%!&%!%8*#'%+,%,8*"'#+*1
H!*?%'.!#'&7%&8"(%!&%5!"').#!6%.)&#&'!*")%'+%!*%!*'#5#+'#"7%!.)%-+F).*)\$%5?%&#*-6)%-)*)&1
H+&'%'.!#'&I&8"(%!&%'()%"+6+.%+,%!%.+&)7%+.%'()%&(!0)%+,%!%*+&)I!.)%-+F).*)\$%5?%&)F).!6
-)*)&%!"'#*-%#*%"+*").'1
J)*)&%"!*%F!.?%#*%6)*-'(@%K+9)%!.)%+*6?%!%,)/%(8*\$.)\$%5!&)%0!#.&%6+*-L%&+9)
"!*%5)%')*&%+,%'(+8&!*\$&%+,%5!&)%0!#.&%6+*-1%<%:;<%9+6)"86)%9!?%"!..?%,.+9%!
(!*\$,86%'+%'(+8&!*\$&%+,%-)*)&1%<%")667%#*%'8.*7%9!?%"+*'!#*%+*)%+.%&)F).!6%:;<
9+6)"86)&%M"(.+9+&+9)&N1%2(8&%'()%*895).%+,%-)*)&%#*%!%")66%"!*%F!.?%-.)!'6?1%!"
#\$%&7%!%5!"').#897%"+*'!#*&%+*)%:;<%9+6)"86)%/#'(%!5+8'%O7PPP%-)*)&%+*%#'1%<
(89!*%")66%"+*'!#*&%43%:;<%9+6)"86)&%"!..?#*-%!%'+'!6%+,%!5+8'%QPP7PPP%-)*)&1
<66%-)*)&%#*%!%-#F)*%")66%!.)%*/' "+0#)\$%#*'+%A;<%M&"'" R)S0.)&&)\$TN%!'%'()%&!9)%'#9)
+.%!'%'()%&!9)%.!')1%2(8&7%/()*%&0)!G#*-%+,%-)*)%,8*"'#+*7%+*)%.),).&%'+%#'&%)S0.)&&#+*
6)F)61%2(#&%.!')%"!*%5)%"+*'.+66)\$%5?%'()%")667%!""+.\$#*-%'+%0.)\$)').9#*)\$%.86)&%/(#"(%!.)
'()9&)6F)&%/.#'')*%#*'+%'()%:;<1
<*%)S!906)@%2()%")66&%#*%+8.%5+\$#)&%M!66%QPP%'.#66#+*%+,%'()9N%)!"(%"+*'!#*%#\$)*'#"!6
:;<%9+6)"86)&1%U)'%6#F).%")66&7%,+.%)S!906)7%)S0.)&&%+*6?%'(+&)%-)*)&%.)=8#.)\$%,+.%6#F).
,8*"'#+*7%/().)!&%&G#*%")66&%)S0.)&&%!%=8#')%\$#,,).)*'%&85&)'%+,%-)*)&1
456\$1,*\$7&\$18'\$"*'/\$."&1&2\$!"'+\$(&2'("1'"/*\$&*9-)&2
A)&'.#"'#+*%)*V?9)&%!.)%0.+')#*&%9!\$)%5?%5!"').#!%!&%!%\$),)*&)%!-!#*&'%,+.)#-*7
#*F!\$#*-%:;<%M,+.%)S!906)7%F#.!6%:;<N1%D!"(%.)&'.#"'#+*%)*V?9)%.)"+-*#V)&%!%8*#=8)
&)=8)*")%+,%'?0#"!66?%4W3%5!&)%0!#.&7%!*\$%/#66%"8'%!*?%:;<%/()*)F).%'(!'%&)=8)*")
+""8.&1
X+.%)S!906)7%'()%.)&'.#"'#+*%)*V?9)%()*Y%>%.)"+-*#V)&%'()%&)=8)*")
MOZ11336:;;11[ZN%!*\$%"8'&%'()%:;<%&'.!*\$%5)'/))*%'()%'/+%3 *8"6)+'#\$)&%#*%'(!'
&)=8)*")1
A)&'.#"'#+*%)*V?9)&%/#66%"8'%:;<%,.+9%!*?%&+8.")7%0.+F#\$)\$%'()%.)"+-*#'#+*%&)=8)*")
#&%0.)&)*'1%>'%\$+)&%*+'%9!'').%#,%'()%:;<%#&%+,%5!"').#!67%06!*'%+.%(89!*%+.#-#*1
<"&1&2\$/0\$456\$1,*\$7&\$=/"*&%\$7-\$456\$#">,2&
:;<%6#-!&)%#&%!*%)*V?9)%'(!'%-68)&%0#)")&%+,%:;<%'+-)'().7%0.+F#\$)\$%'()%)*\$&%!.)E
"+90!'#56)1

58

!"#\$%&'&()*+*&,-&"#.'/&,0&-0,1&,0&2,.'2,&345&+#2&6)2"&!"#7&8&+'/&9*&*'\$):;&<,)/*=&2,
'&()*+*&,-&9'+2*0)':&345&':\$,&+#2&6)2"&!"#7&8>&!")\$&'::,6\$&2"*&+0*'2),/&,-&0*+,.9)/'/2
345\$%&,0 ";90)=\$%&+0*'2*=&9;&<,)/)/1&()*+*\$&,-&345&-0,.&26,&=)--*0*/2&\$,#0+*\$>
?*/*\$&+'/&9*&+#2&,#2&,-&"#.'/&345&,0&(:'/2&345%&'/=&(:'+*=&)/\$)=*&9'+2*0)'>&@,0&
*A'.(:*%&2"*&"#.'/&1*/*&-,0&2"*&",0.,/*&)/\$#:)/&+'/&9*&(#2&)/2,&9'+2*0)'>&B/=*0&2"*&0)1"2&
+,/=)2),/\$%&2"*\$*&9'+2*0)'&+'/&.'C*&'#2"*/2)+&"#.'/&)/\$#:)/>
!"#\$%&'\$(#)*(\$%#""(+&)+,"#)(-&*+*\$(./(012
D:'\$.)=\$&'0*&\$.'::&+)0+#:'0&345\$&-,#/=&)/\$)=*&\$,.*&9'+2*0)':&+*::\$>&!"*;&0*(:)+'2*
2"*)0&,6/&345&9;&9,00,6)/1&2"*&+*::\$E&(,:;.*0'\$*\$>&!"#\$&2"*;&+'/&(*0\$)\$2&)/=*-)/)2*:;
)/\$)=*&+*::\$&6)2",#2&=,)/1&F*0;&.#+"&6,0C&,-&2"*)0&,6/>
G*+'#\$*&,-&2"*)0&\$.'::&\$)H*%&(:'\$.)=&345\$&'0*&*'\$;&2,&*A20'+2&'/=&(#0)-;&-0,.&9'+2*0)':
+*::\$>&I"*/&+#2&6)2"&'&0*\$20)+2),/&*/H;.*%&2"*;&+'/&9*&<,)/*=&2,&-,0*)1/&345\$J-0,.&'/;
\$,#0+*J6")+"&"'F*&9**/&+#2&6)2"&2"*&\$'.*&*/H;.*>
!"*&0*\$#:2)/1&";90)=&345\$&+'/&9*&0*)/20,=#+*=&)/2,&9'+2*0)':&+*::\$&9;&'&(0,+*=#0*
+'::*=&20'/\$-,0.'2),/>&4,6&2"*&";90)=&(:'\$.)=\$&+'/&(*0(*2#'2*&2"*.\$*:F*\$&)/&2"*&9'+2*0)'
<#\$2&'\$&9*-,0*&*A+*(2&2"'2&2"*&-,0*)1/&345&6")+"&6'\$&<,)/*=&2,&)2&)\$&':\$,&9*)/1&(*0(*2#'2*=>
!"*&-,0*)1/&345&1*2\$&'&-0**&0)=*%&\$,&2,&\$(*'C>
KF*0;&";90)=&(:'\$.)=&/,6&+,/2')/\$&'&(*0-*+2&+,(;&,-&2"*&()*+*&,-&-,0*)1/&345&,0)1)/'::;
<,)/*=&2,&)2>&I*&\$';&2"'2&-,0*)1/&()*+*&,-&345&"'\$&9**/&+:,/*=L&2"*&(:'\$.)=&6")+"&+'00)*=
2"*&-,0*)1/&345&)\$&+'::*=&'&+:,/)/1&F*")+:*&,0&F*+2,0>
8/&'==)2),/&2,&2"*)0&#\$*-#:/*\$\$&-,0&+:,/)/1&-,0*)1/&1*/*\$%&(:'\$.)=\$&\$,.*2).*\$&+'00;
1*/*\$&,-&2"*)0&,6/>&G'+2*0)'&=)*&6"*/&*A(,\$*=&2,&'/2)9),2)+\$>&7,6*F*0%&'/2)9),2)+M0*\$)\$M
2'/+*&1*/*\$&'::,6&9'+2*0)'&2,&10,6&)/&2"*&(0*\$*/+*&,-&'/&'/2)9),2)+&\$#+"&'\$&'.()+)::)/>&N#+"
1*/*\$&'0*&,-2*/&-,#/=&,/&(:'\$.)=\$>&I"*/&-,0*)1/&345&)\$&)/\$*02*=&)/2,&\$#+"&(:'\$.)=\$%&'/=
2"*&";90)=\$&)/20,=#+*=&)/2,&9'+2*0)':&+*::\$&9;&20'/\$-,0.'2),/%&)2&)\$&*'\$;&2,&\$*:*+2&2",\$*&
9'+2*0)'&2"'2&"'F*&0*+*)F*=&2"*&(:'\$.)=J9*+'#\$*&2"*;&"'F*&'+O#)0*=&2"*&'9):)2;&2,&10,6&)/
2"*&(0*\$*/+*&,-&2"*&'/2)9),2)+%&6"*0*'\$&'::&,2"*0&9'+2*0)':&+*::\$&'0*&C)::*=>
012("&3)#)&*\$
I"*/&345&)\$&*A20'+2*=&-0,.&'&1)F*/&+*::&2;(*%&)2&+'/&9*&+#2&)/2,&()*+*\$&'/=&2"*&()*+*\$
+'/&9*&+:,/*=&*/&.'\$\$*&)/2,&'&(,(#:'2),/&,-&(:'\$.)=\$>&!")\$&(0,+*\$\$&(0,=#+*\$&'&(,(#:'2),/
,-&";90)=&P0*+,.9)/'/2Q&345\$>&5-2*0&)/20,=#+)/1&2"*\$*&";90)=\$&9'+C&)/2,&+*::\$%&*'+"
20'/\$-,0.*=&+*::&6)::&"'F*&0*+*)F*=&'/=&(0,('1'2*=&,/*&#/)O#*&";90)=>&KF*0;&";90)=&6)::
+,/2')/&2"*&\$'.*&F*+2,0&345&9#2&'&=)--*0*/2&)/\$*02&345>
8-&2"*0*&'0*&R%SSS&=)--*0*/2&345&.,:*+#:*\$&)/&2"*&,0)1)/':&.)A2#0*%&R%SSS&=)--*0*/2
";90)=\$&6)::&9*&-,0.*=L&R%SSS&=)--*0*/2&20'/\$-,0.'/2&+*::\$&6)::&9*&0*+,F*0*=%&*'+"&+'00;)/1
,/*&,-&2"*&,0)1)/':&R%SSS&()*+*\$&,-&1*/*2)+&)/-,0.'2),/>&N#+"&'&+,::*+2),/&)\$&+'::*=&'&345
:)90'0;>&8-&2"*&,0)1)/':&*A20'+2&+'.*&-0,.&"#.'/&+*::\$%&2"*&:)90'0;&)\$&'&"#.'/&:)90'0;>
8/=)F)=#':&345\$&,-&)/2*0*\$2&+'/&9*&-)\$"*=&,#2&,-&\$#+"&'&:)90'0;&9;&\$+0**/)/1&2"*&:)90'0;
6)2"&'/&'((0,(0)'2*&(0,9*>

59

## Appendix D Gene Regulation

!"#\$%&'()*\$+&,-.(,\$-/&"*.,'*\$&0\$'(00)#),-\$1#&-)(,*\$2/(+/\$1)#0&#3\$3.,4\$'(00)#),-\$5&%*6
7(8)*-(9)\$),:43)*\$.#)\$1#&-)(,*;\$*&3)\$&0\$-/)\$/&#3&,)\$*(8,.<*\$-/.-\$#",\$-/#&"8/\$&"#\$%&'()*
.,'\$-/)\$.,-(%&'()*\$1#&-)+-(,8\$"*\$0#&3\$'(*).*)\$.#)\$1#&-)(,*6\$=/)\$(,0&#3.-(&,\$0&#\$.**)3%<(,8
.\$1#&-)(,\$(*\$+.##()'\$(,\$&"#\$7>?6\$=/)\$*)+-(&,\$&0\$7>?\$2/(+/\$+&,-.(,*\$-/)\$+&')\$0&#\$3.@(,8\$.
1#&-)(,\$(*\$+.<<)'\$.\$8),)6\$=/)#)\$.#)\$&9)#\$ABCBBBDEBBCBBB\$8),)*\$(,\$-/)\$/"3.,\$8),&3)6\$F.+/
8),)\$+&')*\$0&#\$.\$",(G")\$1#&-)(,H\$&,)\$8),)C\$&,)\$1#&-)(,6\$=/)\$8),)\$-/.-\$+&')*\$0&#\$.\$'(8)*-(9)
),:43)\$(,\$4&"#\$3&"-/\$(*\$'(00)#),-\$0#&3\$&,)\$-/.-\$+&')*\$0&#\$.,\$.,-(%&'4\$&#\$-/)\$1(83),-\$-/.+&<&#*\$4&"#\$)4)*6\$\$
!#8.,(*3*\$#)8"<.-)\$)I1#)**(&,\$&0\$-/)(#\$8),)*\$.,'\$"<-(3.-)<4\$-/)\$.3&",-*\$.,'\$@(,'*\$&0
1#&-)(,*\$1#)*),-\$2(-/(,\$-/)(#\$+)<<*\$0&#\$.\$34#(.'\$&0\$#).*&,*C\$(,+<"'(,8\$')9)<&13),-.<\$+/.,8)*C
+)<<"<.#\$*1)+(.<(:.-(&,C\$.,'\$.'.1-.-(&,\$-&\$-/)\$),9(#&,3),-6\$J),)\$#)8"<.-(&,\$,&-\$&,<4\$.<<&2*\$0&#
.'.1-.-(&,\$-&\$'(00)#(,8\$+&,'(-(&,*C\$%"-\$.<*&\$1#)9),-*\$2.*-)0"<\$&9)#1#&'"+-(&,\$&0\$",,))')'
1#&-)(,*\$2/(+/\$2&"<'\$1"-\$-/)\$&#8.,(*3\$.-\$.\$+&31)-(-(9)\$'(*.'9.,-.8)6\$=/)\$8),)*\$(,9&<9)'\$(,
-/)\$-#.,*1&#-\$.,'\$%#).@'&2,\$K+.-.%&<(*3L\$&0\$0&&'\$.#)\$8&&'\$)I.31<)*\$&0\$/(8/<4\$#)8"<.-)'
8),)*6\$M&#\$)I.31<)C\$-/)\$*"8.#\$.#.%(,&*)\$(*\$%&-/\$.\$*&"#+)\$&0\$),)#84\$.,'\$.\$*&"#+)\$&0\$+.#%&,6\$!"
#\$%&'%.+-)#(.\$1#&'"+)\$-/#))\$),:43)*\$K1#&-)(,*L\$,))')'\$-&\$'(8)*-\$.#.%(,&*)\$.*\$.\$0&&'\$*&"#+)6
=/)\$8),)*\$2/(+/\$+&')\$0&#\$-/)*)\$),:43)*\$.#)\$,&-\$)I1#)**)'\$2/),\$.#.%(,&*)\$(*\$.%*),-C\$%"-\$-/)4
.#)\$)I1#)**)'\$2/),\$.#.%(,&*)\$(*\$1#)*),-\$(,\$-/)(#\$),9(#&,3),-6\$N&2\$(*\$-/(*\$*&O
P)8"<.-(&,\$&0\$-/)\$)I1#)**(&,\$&0\$1#&-)(,*\$&0-),\$&++"#*\$.-\$-/)\$<)9)<\$&0\$-#.,*+#(1-(&,\$0#&3
7>?\$(,-&\$P>?6\$=/(*\$#)8"<.-(&,\$-.@)*\$1<.+)\$.-\$.\$9)#4\$*1)+(0(+\$<&+.-(&,\$&,\$-/)\$7>?\$-)31<.-)C
+.<<)'\$.\$1#&3&-)#C\$2/)#)\$P>?\$1&<43)#.*)\$*(-*\$'&2,\$&,\$-/)\$7>?\$.,'\$%)8(,*\$-#.,*+#(1-(&,\$&0
-/)\$8),)6\$Q,\$%.+-)#(.C\$8#&"1*\$&0\$#)<.-)'\$8),)*\$.#)\$&0-),\$+<"*-)#)'\$-&8)-/)#\$.,'\$-#.,*+#(%)'\$(,-&
P>?\$0#&3\$&,)\$1#&3&-)#6\$=/)*)\$+<"*-)#*\$&0\$8),)*\$+&,-#&<<)'\$%4\$.\$*(,8<)\$1#&3&-)#\$.#)\$+.<<)'
&1)#&,*6\$\$
=/)\$-/#))\$8),)*\$K()(*+'()(,'.,' ()(-L\$-/.-\$+&')\$0&#\$-/#))\$'(8)*-(9)\$),:43)*\$(,9&<9)'\$(,
-/)\$%#).@'&2,\$&0\$.#.%(,&*)\$.#)\$+<"*-)#)'\$-&8)-/)#\$(,\$2/.-\$(*\$@,&2,\$.*\$-/)\$.#.%(,&*)\$&1)#&,6A
=/)*)\$-/#))\$1#&-)(,*\$.#)\$')1),'),-\$&,\$(,(-(.-(&,\$&0\$-#.,*+#(1-(&,\$0#&3\$.\$*(,8<)\$1#&3&-)#C\$R*,-6
=#.,*+#(1-(&,\$&0\$-/)*)\$-/#))\$8),)*\$#)G"(#)*\$-/)\$*(3"<-.,)&"*\$1#)*),+)\$&0\$-/)\$7>?\$-)31<.-)
K1#&3&-)#\$.,'\$&1)#&,LC\$P>?\$1&<43)#.*)C\$.\$7>?\$%(,'(,8\$1#&-)(,\$+.<<)'\$()(. .,'\$.#.%(,&*)6
()(. %(,'*\$-&\$-/)\$7>?\$.-\$-/)\$%(,'(,8\$*(-)\$0&#\$-/)\$P>?\$1&<43)#.*)\$K-/)\$%)8(,,(,8\$&0\$-/)\$
.#.%(,&*)\$&1)#&,L6\$S/),\$.#.%(,&*)\$(*\$1#)*),-\$(,\$-/)\$),9(#&,3),-C\$%.+-)#(.\$-.@)\$(-\$"16\$!,+)
(,*(')C\$-/)\$.#.%(,&*)\$(,-)#.+-*\$'(#)+-<4\$2(-/\$()(. 2/(+/\$(*\$%&",'\$-&\$-/)\$7>?6\$=/)\$(,-)#.+-(&,
+."*)*\$()(. -&\$+/.,8)\$(-*\$*/.1)\$2/(+/\$(,\$-"#,\$1#&3&-)*\$K.+-".<<4\$/)<1*L\$-/)\$%(,'(,8\$&0\$P>?
1&<43)#.*)\$.,'\$-/)\$-/#))\$8),)*\$()(*C\$, .,'\$-C\$.#)\$-#.,*+#(%)'6\$=/#))\$),:43)*\$.#)\$1#&'"+)'C
-/)4\$%#).@\$'&2,\$.#.%(,&*)C\$.,'\$)9),-".<<4\$-/)\$.#.%(,&*)\$#",*\$&"-6\$Q,\$-/)\$.%*),+)\$&0\$.#.%(,&*)
-/)\$()(T\$#)-"#,*\$-&\$(-*\$&#(8(,.<\$*/.1)\$.,'\$-#.,*+#(1-(&,\$(*\$*/"-\$&006
=/)\$7>?\$+&')\$&0\$-/)\$1JU!\$1<.*3('\$/.*\$%)),\$),8(,))#)'\$-&\$(,+&#1&#.-)\$.*1)+-*\$&0\$-/)
.#.%(,&*)\$&1)#&,6\$V&-/\$-/)\$1#&3&-)#\$KR*,-L\$.,'\$-/)\$()(T\$8),)\$.#)\$1#)*),-6\$N&2)9)#C\$-/)
8),)*\$2/(+/\$+&')\$0&#\$.#.%(,&*)\$+.-.%&<(*3C\$()(*+','.,' -C\$/.9)\$%)),\$#)1<.+)'\$%4\$-/)\$*(,8<)
8),)\$2/(+/\$+&')*\$0&#\$JMR6\$=/)#)0&#)C\$(,\$-/)\$1#)*),+)\$&0\$.#.%(,&*)C\$()(. 1#&-)(,\$1#&3&-)*
-/)\$%(,'(,8\$&0\$P>?\$1&<43)#.*)\$.,'\$JMR\$(*\$1#&'"+)'6\$T)<<*\$0<"&#)*+)\$%#(<<(.,-\$8#)),\$.*\$-/)4
1#&'"+)\$3&#)\$.,'\$3&#)\$JMR6\$Q,\$-/)\$.%*),+)\$&0\$.#.%(,&*)C\$()(. ,&\$<&,8)#\$0.+(<(-.-)*\$-/)
%(,'(,8\$&0\$P>?\$1&<43)#.*)\$.,'\$-/)\$JMR\$8),)\$(*\$,&-\$-#.,*+#(%)'6\$S/),\$JMR\$(*\$,&-\$3.')C
%.+-)#(.\$+&<&,()*\$2(<<\$.11).#\$-&\$/.9)\$.\$2(<'W-41)\$K,.-"#.<L\$1/),&-41)X&0\$2/(-)\$+&<&,()*
2(-/\$,&\$0<"&#)*+),+)6\$\$
=/(*\$(*\$.,\$)I+)<<),-\$)I.31<)\$&0\$-/)\$+),-#.<\$3&<)+"<.#\$0#.3)2&#@\$&0\$%(&<&84\$(,\$.+-(&,H\$\$
7>?P>?RP!=FQ>=P?Q=6

60

araC

araB

araA

araA

araA

arabinose

araB
araC
RNA Pol

RNA Pol

araB
araC

m RNA

araB

RNA Pol

araC

## Expression of Green Fluorescent Protein

araC

GFP

arabinose

GFP
araC
RNA Pol

RNA Pol

GFP
araC

m RNA

GFP
araC

61

RNA Pol

Appendix E References
!"
@"
>"

#\$%\$&\$%'()*+,-\$.'(/0+123.(*%(04\$%.5*46\$02*%(*5(!"#\$%&'#\$'()#*+' 720&(8-\$.621."(,-).*+-)/'*+"'(!""'
99:(;!<=>?"((
#\$%\$&\$%'()*+,-\$.'(A3B&%2C+3.(5*4(04\$%.5*46\$02*%(*5(!-)#*+'"(D%()EF(G-*%2%,H(F(I4\$B02B\$-(F884*\$B&
;J1"()"(K"(L-*M34?'(M*-"(!"(DNO(I43..'(PQ5*41(;!<=:?"
/B&-325'(N*R340'A7*(8*.202M3-S(43,+-\$031(.S.036.'((&( \$%1(0(+'(D%(!"#\$%&'#\$'()#*+')\$%1 1(+0*2%++('
G3--+-\$4(\$%1(K*-3B+-\$4(T2*-*,S'(E321&\$410"(F/K(I43..'(U\$.&2%,0*%'()"G"(;!<<V?"

I\$4\$52-6(2.(\$(04\$136\$4W(*5(F6342B\$%(E\$02*%\$-(G\$%(G*"
X3--YP(2.(\$(04\$136\$4W(*5(Z4\$50([**1.'(D%B"

62